Method for producing cartilage cells induced to be differentiated from stem cells

ABSTRACT

The present invention relates to a method for inducing differentiation, into chondrocytes, of cord blood mononuclear cell-derived induced pluripotent stem cells. In a case where a chondrogenic pellet produced by the method of the present invention is transplanted into a cartilage damage area in vivo, regeneration of cartilage can be effectively exhibited by differentiated chondrocytes. In such a case, an effective cartilage regeneration capacity can be exhibited as compared with a case where chondrocytes produced by differentiation induction with the addition of a recombinant growth factor are transplanted. Thus, the present invention can be usefully used for tissue engineering therapies.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. Ser. No. 16/479,079,filed Jul. 18, 2019, which is a 35 U.S.C. 371 National Phase Entry fromPCT/KR2018/000903, filed Jan. 19, 2018, which claims priority to KoreanPatent Application No. 10-2017-0009015 filed on Jan. 19, 2017, andKorean Patent Application No. 10-2017-0009019 filed on Jan. 19, 2017,the entireties of which are incorporated herein by reference.

This research was supported by a grant of the Korean Health TechnologyR&D Project through the Korea Health Industry Development Institute(KHIDI), funded by Ministry of Health & Welfare, Republic of Korea(HH6C2177, HI8C1178, HO16C0001).

SEQUENCE LISTING STATEMENT

The instant application contains a Sequence Listing in electronic formatwhich has been submitted via EFS-Web. Said Sequence Listing, created onFeb. 8, 2023, is named “4669-126US2_ST26.XML” and is 8192 bytes in size.The information in the electronic format of the Sequence Listing is partof the present application and is incorporated herein by reference inits entirety.

TECHNICAL FIELD

The present invention relates to a method for producing a compositionfor differentiation into chondrocytes, a chondrocyte produced bydifferentiation therefrom, and a pharmaceutical composition forpreventing or treating a cartilage-related disease, comprising thecomposition for differentiation into chondrocytes or the chondrocyteproduced by differentiation, which are capable of providing newstrategies for personalized regenerative medicine.

BACKGROUND

Cartilage is a bone tissue composed of chondrocytes and cartilagematrix, and usually refers to a tissue that forms part of a joint.Cartilage consists mostly of chondrocytes and a large amount ofextracellular matrix (ECM) in which various types of collagen,proteoglycans, and flexible fibers are enriched. As a result, cartilagehas high elasticity and a very low coefficient of friction, and servesas a buffer to prevent friction between the ends of bone, therebyhelping joints to move with little friction. In addition, cartilageserves to construct a framework for a portion that requires elasticity,such as an organ of the respiratory tract or auricle, or a portion thatrequires resistance to pressure, such as costal cartilage or symphysealcartilage.

In particular, articular cartilage is composed of chondrocytes that arecells specially differentiated so as to be distributed between cartilagematrices. Chondrocytes serve to create and maintain articular cartilage.Cell division occurs in chondroblasts. However, once growth stops,chondrocytes are trapped in a small space called lacuna and no longerdivide in a normal environment.

Therefore, once cartilage is damaged, regeneration thereof is difficultto occur. In addition, since cartilage is an avascular tissue, there isno blood vessel for nutritional supply, so that there is a limitationfor migration of stem cells and regeneration capacity of tissue isdecreased. Therefore, various regenerative medicine researches have beenconducted for the purpose of increasing regeneration capacity ofcartilage, such as inducing differentiation into chondrocytes forregeneration of cartilage. The most commonly used technique for this isa cell therapy technique using cells. However, such a technique has adisadvantage in that proliferation efficiency differs depending on typesof cell lines so that there may be limitations. Accordingly, it hasrecently been known that adult stem cells such as adipocyte-derived stemcells or mesenchymal stem cells (MSCs), which are isolated autologouslyfrom patients, are induced to differentiate into chondrocytes in vitroand used for cell therapies.

However, even in such cell therapies, early chondrocytes and adult stemcells rapidly lose characteristics of chondrocytes when they arecultured in vitro. Thus, there are restrictions that their continued useis limited after being transplanted into the body. In addition, fordifferentiation capacity of adult stem cells to differentiate intochondrocytes, characteristics thereof may vary depending on pathologicalcharacteristics of sources from which the adult stem cells are isolated.Thus, there are concerns about side effects in that many variables maybe present in applying such cell therapies to actual regenerativemedicine.

Accordingly, studies have been attempted to use human inducedpluripotent stem cells (hiPSCs) as cell therapeutic agents for tissueregeneration medicine. The hiPSCs are characterized in that they havedifferentiation capacity to differentiate into various lineages and thuscan be used regardless of types of tissues in tissue regenerationmedicine [NPL 1]. Therefore, even for a tissue in which lowdifferentiation capacity is exhibited in a case of being induced todifferentiate from stem cells, a relatively high regeneration effect canbe expected in a case of using hiPSCs.

Among growth factors used for inducing hiPSCs to differentiate intochondrocytes, bone morphogenetic protein (BMP), transforming growthfactor (TGF), or the like has recently been used [NPL 2]. BMP alone isknown to play a major role in development of bone and cartilage, and isalso known to affect the survival and proliferation of chondrocytes.Among these, it has been reported that due to deficiency of BMP2,development of endochondral bone may be inhibited [NPL 3].

TGFβ proteins are known as factors that regulate structural frameworksin various tissues through their involvement in processes such asproliferation, differentiation, and death of cells. The TGFβ proteinsare divided into three protein isoforms such as TGFβ1, TGFβ2, and TGFβ3,and it has been reported that cartilage formation may be induced throughaction among these isoforms [NPLS 4 and 5]. Among these, cartilageformation may be induced, in particular, using TGFβ1 or TGFβ3, in whichTGFβ3 has been reported to have higher differentiation capacity [NPL 6].In the process of inducing stem cells to differentiate into cells of atarget tissue, use of recombinant human growth factors may function asan important element. However, this requires frequent addition of growthfactor molecules during differentiation, which involves a disadvantagethat an expensive cost is incurred.

In order to overcome such a disadvantage, attempts have been made, inthe process of differentiating stem cells, to induce differentiationthereof into cells of a target tissue by inducing overexpression ofgrowth factors through gene transfer in a state where growth factors arenot added to a medium. With regard to differentiation into chondrocytes,it has been reported that in a case where gene delivery of TGFβ1 is madewith a viral vector in synovial-derived MSCs, cell proliferation may beincreased and a rate of chondrogenic differentiation may be accelerated[NPL 7]. In addition, in a case where SOX9 is overexpressed in mousemesenchymal stem cells (MSCs) and cord blood-derived MSCs,differentiation into chondrocytes may be promoted [NPLS 8 and 9], andexpression of type 2 collagen may be also increased [NPL 10].

With regard to gene delivery techniques for such cell therapies, anon-viral gene delivery technique has attracted attention as a safemethod. This is because commercial DNA vector plasmids contain sequencesof bacterial origin, which may induce an immune response to bacterialproteins. For this purpose, a minicircle vector can be used, and theminicircle vector refers to a vector having a relatively small size,obtained by removing an antibiotic resistance gene, a gene encoding abacterial structural protein, and a transcription unit gene. Theminicircle vector has a feature capable of expressing an exogenous geneat a high level in vitro and in vivo, along with advantages of beingsmall in size and capable of avoiding an immune response. Thus, theminicircle vector attracts attention for its potential of beingbeneficially used in preclinical gene therapy studies [NPL 11]. Throughthis, safe and efficient gene delivery becomes possible, and thus atherapeutic effect can be enhanced in a case where genetically modifiedstem cells are applied to therapies [NPL 12].

In addition, in conventional methods for inducing differentiation ofstem cells into chondrocytes, differentiation into chondrocytes isinduced in a process in which induced pluripotent stem cell-derivedembryoid bodies are generated and differentiation into chondrocytes iscontinuously induced therefrom. In this process, in order to increasedifferentiation efficiency, a method has been studied in which embryoidbodies are cultured in a gelatin medium to obtain outgrowth cells (OGcells), and the resulting cells are caused to differentiate into singlecells so that differentiated chondrocytes are obtained. However, sinceit is required to further increase differentiation efficiency so as touse chondrocytes, which are produced by differentiation induction fromstem cells, as a cell therapeutic agent in regenerative medicine,studies on development of a method for more effectively producingchondrocytes have been continuously conducted.

Accordingly, the present inventors have studied to develop a method inwhich stem cells are induced to differentiate into a chondrogenic pelletand the chondrogenic pellet is effectively applied to cartilageregeneration therapies. As a result, first, the present inventors haveidentified that in a case where minicircle vectors expressing the growthfactors BMP2 and TGFβ3 are constructed and transduced into stem cells,and the stem cells are induced to differentiate into chondrocytes, thestem cells can effectively differentiate into a chondrogenic pellet. Thepresent inventors have identified that the differentiated chondrogenicpellet can significantly express chondrocyte marker genes and canexhibit a significant cartilage regeneration effect in an osteochondraldefect area in a case of being transplanted into the living body.

In addition, the present inventors have identified that in a case whereiPSCs are cultured to obtain embryoid bodies, outgrowth cells (OG cells)are produced therefrom, and then the OG cells are isolated by sizesthrough centrifugation, as the OG cells are smaller in size, higherefficiency of differentiation into a chondrogenic pellet is exhibited,thereby having completed the present invention.

In addition, there is therefore a need for an alternative system thatcan cover a large portion of the population with minimal cell lines.This alternative system should have a low risk of allograft rejection ina case of being used in many patients. The history of successfultransplantation of organs and hematopoietic stem cells has establishedimportance of HLA genes, which is directly related to the individual'simmune identity. Banking of ESCs isolated from HLA-homozygousindividuals was recommended in the year 2005 to decrease the number ofrequired cell lines. This banking system exclusively requires cells,which are homozygous for only one of HLA-A, -B and -DRB1 haplotypes,from donors. These three subsets of genes are most important in the HLAloci and to decrease possibility of rejection. This strategy was appliedto settlement of iPSC banking.

Accordingly, the present inventors have classified the homozygous HLA-A,-B, and -DRB1 types that can maximally cover the (South) Koreanpopulation using data from the Catholic Hematopoietic Stem Cell Bank.CBMCs and PBMCs were obtained from donors to produce iPSCs at a clinicallevel and reprogrammed according to the Good Manufacturing Practice. Onthe basis of this, the present invention includes a report on the firsthomozygous iPS cell line in Korea, the report having been made, underthe sponsorship of the Korean government, for research and clinicaltrial.

CITATION LIST Non-Patent Literature

-   [NPL 1] Kim Y, Rim Y A, Yi H, Park N, Park S H, Ju J H: The    Generation of Human Induced Pluripotent Stem Cells from Blood Cells:    An Efficient Protocol Using Serial Plating of Reprogrammed Cells by    Centrifugation. Stem Cells Int 2016, 2016:1329459.-   [NPL 2] Nam Y, Rim Y A, Jung S M, Ju J H: Cord blood cell-derived    iPSCs as a new candidate for chondrogenic differentiation and    cartilage regeneration. Stem Cell Res Ther 2017, 8:16.-   [NPL 3] Shu B, Zhang M, Xie R, Wang M, Jin H, Hou W, Tang D, Harris    S E, Mishina Y, O'Keefe R J, et al: BMP2, but not BMP4, is crucial    for chondrocyte proliferation and maturation during endochondral    bone development. J Cell Sci 2011, 124:3428-3440.-   [NPL 4] Tuli R, Tuli S, Nandi S, Huang X, Manner P A, Hozack W J,    Danielson K G, Hall D J, Tuan R S: Transforming growth    factor-beta-mediated chondrogenesis of human mesenchymal progenitor    cells involves N-cadherin and mitogen-activated protein kinase and    Wnt signaling cross-talk. J Biol Chem 2003, 278:41227-41236.-   [NPL 5] Mueller M B, Tuan R S: Functional characterization of    hypertrophy in chondrogenesis of human mesenchymal stem cells.    Arthritis Rheum 2008, 58:1377-1388.-   [NPL 6] Barry F, Boynton R E, Liu B, Murphy J M: Chondrogenic    differentiation of mesenchymal stem cells from bone marrow:    differentiation-dependent gene expression of matrix components. Exp    Cell Res 2001, 268:189-200.-   [NPL 7] Kim Y I, Ryu J S, Yeo J E, Choi Y J, Kim Y S, Ko K, Koh Y G:    Overexpression of TGF-beta1 enhances chondrogenic differentiation    and proliferation of human synovium-derived stem cells. Biochem    Biophys Res Commun 2014, 450:1593-1599.-   [NPL 8] Tsuchiya H, Kitoh H, Sugiura F, Ishiguro N: Chondrogenesis    enhanced by overexpression of sox9 gene in mouse bone marrow-derived    mesenchymal stem cells. Biochem Biophys Res Commun 2003,    301:338-343.-   [NPL 9] Wang Z H, Li X L, He X J, Wu B J, Xu M, Chang H M, Zhang X    H, Xing Z, Jing X H, Kong D M, et al: Delivery of the Sox9 gene    promotes chondrogenic differentiation of human umbilical cord    blood-derived mesenchymal stem cells in an in vitro model. Braz J    Med Biol Res 2014, 47:279-286.-   [NPL 10] Kim J H, Do H J, Yang H M, Oh J H, Choi S J, Kim D K, Cha K    Y, Chung H M: Overexpression of SOX9 in mouse embryonic stem cells    directs the immediate chondrogenic commitment. Exp Mol Med 2005,    37:261-268.-   [NPL 11] Gill D R, Pringle I A, Hyde S C: Progress and prospects:    the design and production of plasmid vectors. Gene Ther 2009,    16:165-171.-   [NPL 12] Bandara N, Gurusinghe S, Chen H, Chen S, Wang L X, Lim S Y,    Strappe P: Minicircle DNA-mediated endothelial nitric oxide synthase    gene transfer enhances angiogenic responses of bone marrow-derived    mesenchymal stem cells. Stem Cell Res Ther 2016, 7:48.

SUMMARY

As described above, since cartilage is difficult to regenerate, aneffective regenerative treatment method is required. Accordingly, anobject of the present invention is to provide a chondrocyte obtained bydifferentiation induction from stem cells and a method for producing thesame.

In addition, another object of the present invention is to provide apharmaceutical composition for preventing or treating a cartilage defectdisease, comprising, as an active ingredient, the chondrocyte obtainedby differentiation induction.

In order to achieve the above objects, the present invention provides,as a first technical solution, a method for producing chondrocytesobtained by differentiation induction from stem cells, comprising thefollowing steps i) to v):

-   -   i) culturing induced pluripotent stem cells (iPSCs) to generate        embryoid bodies (EBs);    -   ii) culturing the EBs generated in step i) in a gelatin-coated        medium, to obtain outgrowth cells (OG cells);    -   iii) transducing the OG cells obtained in step ii) with either        or both of a minicircle vector that contains a base sequence        encoding BMP2 and a minicircle vector that contains a base        sequence encoding TGFβ3; and    -   iv) inducing differentiation of the OG cells transduced in        step iii) into chondrocytes;    -   v) obtaining the chondrocytes produced by differentiation        induction in step iv).

In addition, the present invention provides a method for producingchondrocytes obtained by differentiation induction from stem cells,comprising the steps of i) to vi):

-   -   i) culturing iPSCs to generate EBs;    -   ii) culturing the EBs generated in step i) in a gelatin-coated        medium, to obtain OG cells;    -   iii) transducing the OG cells obtained in step ii) with a        minicircle vector that contains a base sequence encoding BMP2;    -   iv) transducing the OG cells obtained in step ii) with a        minicircle vector that contains a base sequence encoding TGFβ3;    -   v) performing mixed culture of the OG cells transduced in        step iii) and the OG cells transduced in step iv), so that the        OG cells are induced to differentiate into chondrocytes; and    -   vi) obtaining the chondrocytes produced by differentiation        induction in step v).

In a preferred embodiment of the present invention, the minicirclevector that contains a base sequence encoding BMP2 may be a non-viralvector, the non-viral vector, (a) containing a gene expression cassettethat contains a CMV promoter, a BMP2 gene consisting of the basesequence of SEQ ID NO: 1, and an SV40 polyadenylation sequence; (b)containing the att attachment sequence of bacteriophage lambda, locatedoutside the gene expression cassette of (a); and (c) not containing areplication origin and an antibiotic resistance gene.

In a further preferred embodiment of the present invention, theminicircle vector that contains a base sequence encoding TGFβ3 may be anon-viral vector, the non-viral vector, (a) containing a gene expressioncassette that contains a CMV promoter, a TGFβ3 gene consisting of thebase sequence of SEQ ID NO: 2, and an SV40 polyadenylation sequence; (b)containing the att attachment sequence of bacteriophage lambda, locatedoutside the gene expression cassette of (a); and (c) not containing areplication origin and an antibiotic resistance gene.

In addition, in a further preferred embodiment of the present invention,the step of inducing differentiation of the OG cells into chondrocytesmay be performed by culturing the cells in a medium containing norecombinant growth factor for 3 to 30 days.

In addition, the present invention provides, as a second technicalsolution, a method for producing chondrocytes obtained bydifferentiation induction from stem cells, comprising the followingsteps i) to v):

-   -   i) culturing induced pluripotent stem cells (iPSCs) to obtain        embryoid bodies;    -   ii) performing adherent culture of the embryoid bodies obtained        in step i), to obtain outgrowth cells (OG cells);    -   iii) performing centrifugation of the OG cells obtained in        step ii) so that the cells are isolated by sizes, and selecting        light cells;    -   iv) inducing differentiation of the light cells selected in        step iii) into chondrocytes; and    -   v) obtaining the chondrocytes produced by differentiation        induction in step iv).

In a preferred embodiment of the present invention, the inducedpluripotent stem cells of step i) may be obtained by reprogramming cordblood mononuclear cells.

In a preferred embodiment of the invention, the adherent culture in stepii) may be performed by culturing the cells on a gelatin-coated plate.

In a preferred embodiment of the present invention, the centrifugationand selection in step iii) may be performed through the following stepsa) to c):

-   -   a) centrifuging a medium containing the outgrowth cells at 300        rpm to 800 rpm for 3 to 10 seconds, to classify the precipitated        cells as heavy cells;    -   b) centrifuging the supernatant after centrifugation in step a)        at 800 rpm to 1,200 rpm for 3 to 10 seconds, to classify the        precipitated cells as medium cells; and    -   c) centrifuging the supernatant after centrifugation in step b)        at 1,200 rpm to 2,000 rpm for 3 to 10 seconds, to classify the        precipitated cells as light cells.

In a preferred embodiment of the present invention, the differentiationinduction in step iv) may be carried out in a medium containing humanbone morphogenetic protein 2 and human transforming growth factor beta3, in which the medium may additionally be supplemented with an IGF2inhibitor.

In addition, the present invention provides, as a third technicalsolution, a method for producing chondrocytes obtained bydifferentiation induction from stem cells, comprising the steps of:

-   -   i) generating and obtaining embryoid bodies from cord blood        mononuclear cell-derived human induced pluripotent stem cells        (CBMC-hiPSCs);    -   ii) generating and obtaining outgrowth cells from the embryoid        bodies of step i); and    -   iii) culturing the outgrowth cells of step ii) to obtain a        chondrogenic pellet.

According to a preferred embodiment of the present invention, theCBMC-hiPSCs of step i) may be obtained by reprogramming cord bloodmononuclear cells.

According to a further preferred embodiment of the present invention,the outgrowth cells of step ii) may be generated by inoculating theembryoid bodies into a gelatin medium.

According to a further preferred embodiment of the present invention,the embryoid bodies may be inoculated at 50 to 70 per cm 2 of thegelatin medium.

According to a further preferred embodiment of the present invention,the cord blood mononuclear cell-derived human induced pluripotent stemcells in step i) may be HLA homozygotes.

According to a further preferred embodiment of the present invention,the HLA homozygote may have an HLA homozygous type of Korean.

According to a further preferred embodiment of the present invention,the HLA homozygous type of Korean may be any one selected from the groupconsisting of HLA-A*33, HLA-B*44, and HLA-DRB1*13.

In addition, the present invention provides a chondrocyte produced byany one of the above methods.

According to a preferred embodiment of the present invention, thecomposition for differentiation into chondrocytes may be such thatexpression of at least one gene selected from the group consisting ofACAN, COL2A1, COMP, and SOX9 is increased.

According to a further preferred embodiment of the present invention,the composition for differentiation into chondrocytes may be such thatthe gene expression level of COL1A1 or COL10 is lower than the geneexpression level of COL2A1.

In addition, the present invention provides a pharmaceutical compositionfor preventing or treating a cartilage damage disease, comprising thechondrocyte as an active ingredient.

In a preferred embodiment of the present invention, the cartilage damagedisease may be degenerative arthritis, rheumatoid arthritis, fracture,plantar fasciitis, humerus epicondylitis, calcified myositis, nonunionof fracture, or joint injury caused by trauma.

Advantageous Effects of Invention

Accordingly, the present invention provides a chondrogenic pelletdifferentiated from induced pluripotent stem cells (iPSCs) withtransduction of minicircle vectors encoding the growth factors BMP2 andTGFβ3. The chondrogenic pellet significantly expresses chondrocytemarker genes, in which the chondrocyte marker genes can be expressed ata higher level than in the chondrocytes produced by differentiationinduction in a medium supplemented with recombinant growth factors.

In addition, the present invention provides a method for producingchondrocytes, in which outgrowth cells (OG cells) produced from iPSCsare isolated into single unit cells and isolated by sizes throughcentrifugation, and among these, light OG cells are selected and inducedto differentiate into a chondrogenic pellet. In a case where light OGcells are selected and induced to differentiate into a chondrogenicpellet according to the method of the present invention, not onlysignificantly high expression levels of chondrocyte markers are observedas compared with a chondrogenic pellet derived from heavy OG cells, butalso a chondrogenic pellet having a histologically stable structure canbe generated.

The chondrogenic pellet produced according to the method of the presentinvention significantly expresses chondrocyte marker genes, in which thechondrocyte marker genes can be expressed at a higher level than in thechondrocytes produced by differentiation induction in a mediumsupplemented with recombinant growth factors.

In a case where the chondrogenic pellet is transplanted into a cartilagedamage area in the living body, cartilage regeneration may beeffectively exhibited by the differentiated chondrocytes, and effectivecartilage regeneration capacity may be exhibited as compared with a casewhere chondrocytes produced by differentiation induction with theaddition of recombinant growth factors are transplanted. Thus, thechondrogenic pellet can be usefully used for tissue engineeringtherapies for cartilage regeneration.

In addition, the chondrogenic pellet produced according to the method ofthe present invention is produced by differentiation induction fromCBMC-derived iPSCs. The chondrogenic pellet is an HLA homozygote and hashigh expression levels of chondrocyte marker genes having types suitablefor cartilage transplantation, so that the chondrogenic pellet can beusefully used for preventing, ameliorating, or treating acartilage-related disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D illustrate results for characteristics of three CBMC-hiPSClines:

FIG. 1A illustrates morphological results for the generated CBMC-hiPSClines;

FIG. 1B illustrates results showing that the CBMC-hiPSCs are stainedwith alkaline phosphatase;

FIG. 1C illustrates results of relative expression of pluripotencymarkers in the CBMC-hiPSC lines; and

FIG. 1D illustrates results represented by images showingimmunofluorescence staining of the generated CBMC-hiPSC lines.

FIGS. 2A-2E illustrate results of chondrogenic pellet generation usingCBMC-hiPSCs:

FIG. 2A illustrates a schematic diagram of chondrogenic pelletgeneration;

FIG. 2B illustrates a morphological result for CBMC-hiPSCs;

FIG. 2C illustrates a morphological result for the generated EBs;

FIG. 2D illustrates a result represented by an image of outgrowth cellsderived from EBs attached to a gelatin-coated culture dish;

FIG. 2E illustrates a result showing an image of the chondrogenicpellet; and

FIG. 2F illustrates a result represented by a side image of outgrowthcells derived from EBs attached to a gelatin-coated culture dish.

FIG. 3 illustrates results showing, as a genetic characteristic of achondrogenic pellet generated from CBMC-hiPSCs, expression levels ofCOL2A1, ACAN, COMP, and SOX9 in the chondrogenic pellet for 10 days, 20days, and 30 days (*, +p<0.05, **, ++p<0.01, ***, +++p<0.001).

FIG. 4 illustrates results obtained by performing histological analysisof a chondrogenic pellet derived from CBMC-hiPSCs, the results showingimages of the chondrogenic pellet stained with safranin O, alcian blue,and toluidine blue at day 10, day 20, and day 30.

FIGS. 5A-5B illustrate results obtained by performing immunohistologicalanalysis of a chondrogenic pellet derived from BMC-hiPSCs:

FIG. 5A illustrates results showing images of the chondrogenic pelletwhich is stained with antibodies against type 2 collagen and aggrecan,and is harvested at various time points; and

FIG. 5B illustrates results showing images of the chondrogenic pelletstained with antibodies against type 1 collagen.

FIGS. 6A-6B illustrate results obtained by performing further analysisfor genetic markers of chondrogenic pellets derived from CBMC-hiPSCs andMSCs (*, +p<0.05, **, ++p<0.01, ***, +++p<0.001):

FIG. 6A illustrates results showing expression levels of COL1A1, whichis a gene representative of fibroid cartilage, and COL10, which is ahypertrophy marker, at various time points; and

FIG. 6B illustrates results showing ratios of COL2A1 to COL1A1 at day10, day 20, and day 30.

FIG. 7 illustrates results obtained by comparing expression levels ofACAN and CLO2A1, which are cartilage formation-related factors, inchondrocytes differentiated from CBMC-derived iPSCs of the presentinvention and chondrocytes differentiated from peripheral blood cell(PBMC)-derived iPSCs according to the prior art.

FIGS. 8A-8F illustrate results which identify good manufacturingpractice (GMP)-grade homozygous human leukocyte antigen (HLA)-inducedpluripotent stem cells:

FIG. 8A illustrates a schematic diagram of the entire GMP-grade iPSCgeneration process (in which human leukocyte antigen (HLA) types arescreened, cells having a selected HLA type are transferred to a GMPfacility, the cells are reprogrammed with iPSCs in the facility, thecells are subjected to various assays for analysis of characteristics,and then a cell line that has passed the assays is established);

FIG. 8B illustrates results of immunofluorescence staining of thegenerated homozygous IPSCs;

FIG. 8C illustrates a result obtained by identifying a pluripotencymarker through a reverse transcription polymerase chain reaction;

FIG. 8D illustrates a result showing an image obtained by alkalinephosphatase staining and the number of positive iPSC colonies;

FIG. 8E illustrates a result of normal karyotype for the generatediPSCs; and

FIG. 8F illustrates results obtained by immunofluorescence staining ofiPSCs differentiated into three germ layers.

FIGS. 9A-9B illustrate construction and identification of minicirclevectors encoding human growth factors of the present invention:

FIG. 9A illustrates a schematic diagram showing a construction processof minicircle vectors for expression of BMP2 and TGFβ3; and

FIG. 9B illustrates results which identify sizes of a minicircle vector(mcBGF2) that contains a gene encoding BMP2 and a minicircle vector(mcTGFβ3) that contains a gene encoding TGFβ3.

FIGS. 10A-10D illustrate identification of expression efficiency ofmcBAMP2 or mcTGFβ3 transduced into HEK293T cells:

FIG. 10A illustrates photographs, taken by a fluorescence microscope,which identify an expression level of RFP following transduction ofmcBAMP2 or mcTGFβ3 into HEK293T cells;

FIG. 10B illustrates a result which identifies percentage of cells intowhich mcBAMP2 or mcTGFβ3 has been transduced;

FIG. 10C illustrates a result which compares a relative expression levelof BAMP2 in HEK293T cells into which mcBAMP2 or mcTGFβ3 has beentransduced; and

FIG. 10D illustrates a result which compares a relative expression levelof TGFβ3 in HEK293T cells into which mcBAMP2 or mcTGFβ3 has beentransduced.

FIGS. 11A-11H illustrate a chondrogenic pellet produced bydifferentiation induction from iPSCs in the present invention:

FIG. 11A illustrates a schematic diagram of a process of inducing iPSCsto differentiate into a chondrogenic pellet;

FIGS. 11B to 11E illustrate morphology of iPSC colonies (FIG. 11B),embryoid bodies (FIG. 11C), OG cells obtained by performing adherentculture of EBs on a gelatin container (FIG. 11D), and OG cells beforetransduction (FIG. 11E), in the differentiation induction process; and

FIGS. 11F to 11H illustrate photographs, taken by a fluorescencemicroscope, which identify expression of RFP in OG cells into whichminicircle vectors have been transduced.

FIGS. 12A-12I illustrate identification of mesenchymal stem cell-relatedfactors in OG cells into which the minicircle vectors of the presentinvention have been transduced:

FIG. 12A illustrates percentage of OG cells into which the minicirclevectors have been transduced;

FIGS. 12B to 12F illustrate results obtained by identifying expressionlevels of mesenchymal stem cell marker genes in the OG cells into whichthe minicircle vectors have been transduced; and

FIGS. 12G to 12I illustrate results obtained by performing alizarin redstaining (FIG. 12G), oil red O staining (FIG. 12H), and alcian bluestaining (FIG. 12I) of the OG cells into which the minicircle vectorshave been transduced.

FIGS. 13A-13E illustrate results obtained by identifying characteristicsof a chondrogenic pellet produced by differentiation induction withtransduction of minicircle vectors:

FIG. 13A illustrates morphology of cells 5 days after transduction ofmcBAMP2 or mcTGFβ3; and

FIGS. 13B to 13E illustrate results obtained by identifying expressionlevels of RFP, days (FIG. 13B), 10 days (FIG. 13C), 20 days (FIG. 13D),and 30 days (FIG. 13E) after initiation of differentiation inductionfollowing transduction of mcBAMP2 or mcTGFβ3. Results of GFP werechecked to identify fluorescence intensity which is spontaneouslyexpressed in a three-dimensionally cultured chondrogenic pellet.

FIGS. 14A-14F illustrates results obtained by identifying expressionlevels of chondrocyte marker genes in a chondrogenic pellet produced bydifferentiation with transduction of the minicircle vectors of thepresent invention.

FIGS. 15A-15E illustrate results obtained by identifying characteristicsof a chondrogenic pellet produced by differentiation with transductionof the minicircle vectors of the present invention:

FIGS. 15A to 15C illustrate results obtained by performing alcian bluestaining (FIG. safranin O staining (FIG. 15B), and toluidine bluestaining (FIG. C) of the chondrogenic pellet; and

FIGS. 15D and 15E illustrate results which identify production levels ofcollagen expressed in the chondrogenic pellet.

FIGS. 16A-16E illustrate results obtained by identifying in vivocartilage regeneration capacity of a chondrogenic pellet produced bydifferentiation induction with transduction of minicircle vectors:

FIG. 16A illustrates a schematic diagram showing a process in which thechondrogenic pellet is transplanted into an osteochondral defect modelmouse in order to identify in vivo cartilage regeneration capacitythereof;

FIGS. 16B to 16D illustrate results obtained by transplanting thechondrogenic pellet produced by differentiation induction withtransduction of minicircle vectors, and, after 4 weeks, identifying theosteochondral defect area with alcian blue staining (FIG. 16B),toluidine blue staining (FIG. 16C), and safranin staining (FIG. 16D);and

FIG. 16E illustrates a result obtained by identifying, with the ICRSscore, a regeneration degree of cartilage following transplantation ofthe chondrogenic pellet into which minicircle vectors have beentransduced.

FIG. 17 illustrates a schematic diagram which summarizes the method ofthe present invention for producing a chondrogenic pellet from inducedpluripotent stem cells.

FIGS. 18A-18G illustrate identification of differentiation inductionmarkers in OG cells isolated by sizes:

FIGS. 18A and 18B illustrate results obtained by observing morphology ofheavy OG cells, medium OG cells, and light OG cells, after isolation andin adherent culture;

FIGS. 18C to 18E illustrate results obtained by identifying geneexpression levels of SOX9 and COL10 in the heavy OG cells, the medium OGcells, and the light OG cells; and

FIGS. 18F and 18G illustrate results obtained by identifying proteinexpression levels of SOX9 and COL10 in the heavy OG cells, the medium OGcells, and the light OG cells.

FIGS. 19A-19B illustrate identification of characteristics of OGcell-derived chondrogenic pellets:

FIG. 19A illustrates morphology of chondrogenic pellets produced byinducing differentiation of OG cells, which have been isolated by sizes,by being cultured in a chondrogenic differentiation medium; and

FIG. 19B illustrates results obtained by identifying, throughhistological staining, osteogenic capacity of the obtained chondrogenicpellets.

FIG. 20 illustrates results obtained by identifying difference inexpression levels of markers for chondrogenic pellets depending on sizesof OG cells.

FIG. 21 illustrates results obtained by identifying a proliferationdegree of heavy OG cells after performing culture in an environmenttreated with chromeceptin, an IGF2 inhibitor.

FIG. 22 illustrates results obtained by identifying expression levels ofchondrogenic differentiation markers in a case where differentiation ofheavy OG cells into a chondrogenic pellet is induced with treatment withchromeceptin.

FIGS. 23A-23B illustrates a result obtained by identifying chondrocytemarkers in a chondrogenic pellet produced by differentiation inductionin an IGF2 inhibitor-treated medium. Here, the OG cells used in theexperiment were heavy OG cells isolated by centrifugation. As a normalcontrol, a sample induced to differentiate into a chondrogenic pellet inan environment with no chromeceptin treatment was used:

FIG. 23A illustrates morphology of a chondrogenic pellet produced bydifferentiation induction with treatment with 2 mM chromeceptin; and

FIG. 23B illustrates results obtained by identifying gene expressionlevels of COL2A1 and SOX9 in the thus differentiated chondrogenicpellet.

DETAILED DESCRIPTION

Hereinafter, the present invention will be described in detail.

In the field of regenerative medicine, techniques for inducingdifferentiation into chondrocytes through cell therapy techniques arewidely used for cartilage regeneration. However, a therapeutic effectmay vary depending on characteristics of differentiation capacity andtissue-forming capacity of chondrocytes to be transplanted into thebody. For this purpose, a technique for inducing differentiation intoinduced pluripotent stem cell-derived chondrocytes is required, andstudies for the purpose of improving differentiation efficiency andtissue-forming capacity of differentiated chondrocytes are continuing.

Accordingly, the present invention provides a method for producingchondrocytes obtained by differentiation induction from stem cells,comprising the following steps i) to v):

-   -   i) culturing induced pluripotent stem cells (iPSCs) to generate        embryoid bodies (EBs);    -   ii) culturing the EBs generated in step i) in a gelatin-coated        medium, to obtain outgrowth cells (OG cells);    -   iii) transducing the OG cells obtained in step ii) with either        or both of a minicircle vector that contains a base sequence        encoding BMP2 and a minicircle vector that contains a base        sequence encoding TGFβ3;    -   iv) inducing differentiation of the OG cells transduced in        step iii) into chondrocytes; and    -   v) obtaining the chondrocytes produced by differentiation        induction in step iv).

In addition, the present invention provides a method for producingchondrocytes obtained by differentiation induction from stem cells,comprising the steps of i) to vi):

-   -   i) culturing iPSCs to generate EBs;    -   ii) culturing the EBs generated in step i) in a gelatin-coated        medium, to obtain OG cells;    -   iii) transducing the OG cells obtained in step ii) with a        minicircle vector that contains a base sequence encoding BMP2;    -   iv) transducing the OG cells obtained in step ii) with a        minicircle vector that contains a base sequence encoding TGFβ3;    -   v) performing mixed culture of the OG cells transduced in        step iii) and the OG cells transduced in step iv), so that the        OG cells are induced to differentiate into chondrocytes; and    -   vi) obtaining the chondrocytes produced by differentiation        induction in step v).

In addition, the present invention provides a chondrogenic pelletproduced by any one of the above methods.

In the method for producing chondrocytes of the present invention, theiPSCs in step i) may be derived from patient-derived cells, or may becommercially available. However, in a process of intending to improvebiocompatibility in transplanting the chondrocytes of the presentinvention into an osteochondral defect area, it is more preferable thatthe iPSCs be derived from patient-derived cells.

In the method for producing chondrocytes of the present invention, it ismore preferable that the OG cells obtained in step ii) be isolated andobtained in single unit cells. However, the present invention is notlimited thereto. In order to obtain the cells in single unit cells,embryoid bodies can be isolated using a cell strainer or the like. It ispreferable that OG cells as single cells obtainable in the productionmethod of the present invention represent fibrous morphology similar tomesenchymal stem cells.

In the method for producing chondrocytes of the present invention, it ispreferable that the OG cells into which the minicircle vectors have beentransduced be induced to differentiate into a chondrogenic pellet bybeing cultured in a chondrogenic differentiation medium for 3 to 30days. Specifically, it is more preferable that the differentiationinduction be performed by being cultured for 5 to 20 days. However, thepresent invention is not limited thereto. In the differentiationinduction, it is preferable that the chondrogenic differentiation mediumnot further contain a recombinant growth factor.

Therefore, the chondrogenic pellet produced by differentiation frominduced pluripotent stem cells into which minicircle vectors encodingthe growth factors BMP2 and TGFβ3 have been transduced, which isprovided in the present invention, significantly expresses chondrocytemarker genes, in which the chondrocyte marker genes can be expressed ata higher level than in the chondrocytes produced by differentiationinduction in a medium supplemented with recombinant growth factors.

In a case where the chondrogenic pellet is transplanted into a cartilagedamage area in the living body, cartilage regeneration may beeffectively exhibited by the differentiated chondrocytes, and aneffective cartilage regeneration capacity may be exhibited as comparedwith a case where chondrocytes produced by differentiation inductionwith the addition of recombinant growth factors are transplanted. Thus,the chondrogenic pellet can be usefully used for tissue engineeringtherapies for cartilage regeneration.

Accordingly, the present invention provides a method for producingchondrocytes obtained by differentiation induction from stem cells,comprising the following steps i) to v):

-   -   i) culturing induced pluripotent stem cells (iPSCs) to obtain        embryoid bodies;    -   ii) performing adherent culture of the embryoid bodies obtained        in step i), to obtain outgrowth cells (OG cells);    -   iii) performing centrifugation of the OG cells obtained in        step ii) so that the cells are isolated by sizes, and selecting        light cells;    -   iv) inducing differentiation of the light cells selected in        step iii) into chondrocytes; and    -   v) obtaining the chondrocytes produced by differentiation        induction in step iv).

In addition, the present invention provides a chondrocyte produced bythe above method.

In addition, in the method for producing chondrocytes of the presentinvention, the “centrifugation” in step iii) is carried out with theintention of selecting light outgrowth cells (OG cells). For thispurpose, it is preferable that the OG cells obtained in step ii) be usedin a state of being isolated into single unit cells by removal of cellmasses. As such, in a case of being isolated into single unit cells, itis expectable that the respective cells can be significantly isolated bysizes. In a case of embryoid bodies cultured in an aggregated form, inorder to isolate the same into single unit cells, it is possible toachieve isolation by a conventional method such as using a cellstrainer.

In the method for producing chondrocytes of the present invention, it ispreferable that the “centrifugation” and “selection” in step iii) becarried out through the following steps a) to c):

-   -   a) centrifuging a medium containing the outgrowth cells at 300        rpm to 800 rpm for 3 to 10 seconds, to classify the precipitated        cells as heavy cells;    -   b) centrifuging the supernatant after centrifugation in step a)        at 800 rpm to 1,200 rpm for 3 to 10 seconds, to classify the        precipitated cells as medium cells; and    -   c) centrifuging the supernatant after centrifugation in step b)        at 1,200 rpm to 2,000 rpm for 3 to 10 seconds, to classify the        precipitated cells as light cells.

Specifically, for conditions of the above-mentioned “centrifugation”, itis preferable to perform centrifugation at 500 rpm for 5 seconds in stepa); it is preferable to perform centrifugation at 1,100 rpm for 5seconds in step b); and it is more preferable to perform centrifugationat 1,500 rpm for 5 seconds in step c). However, the present invention isnot limited thereto. In the method of the present invention, from theviewpoint that light OG cells are selected and induced to differentiateinto chondrocytes, modification can be made so that the step a) isomitted and only the step b) is performed, and then a step ofclassifying the cells as light cells is performed through the step c)using the supernatant after centrifugation. However, the presentinvention is not limited thereto, and the method of the presentinvention can be applied without limitation as long as the method is amethod that belongs to a scope which can be understood by a personskilled in the art to be capable of selecting only light cells.

In the method of the present invention, the “induced pluripotent stemcells” of step i) may be derived from patient-derived cells, or may becommercially available. However, from the viewpoint that it is intendedto enhance biocompatibility in transplanting the chondrocytes of thepresent invention into an osteochondral defect area, it is morepreferable to use, as the iPSCs, those induced from the patient-derivedcells. Specifically, it is most preferable to use, as the iPSCs, thoseobtained by reprogramming the patient's cord blood mononuclear cells.However, the present invention is not limited thereto.

In the method of the present invention, the “adhesion culture” in stepii) may be such that the cells are cultured on a gelatin-coated plate.

In the method of the present invention, it is preferable that the“inducing differentiation” in step iv) be performed in a mediumcontaining human bone morphogenetic protein 2 and human transforminggrowth factor beta 3. However, the present invention is not limitedthereto. Differentiation inducing factors known as factors capable ofinducing differentiation of stem cells into chondrocytes may beoptionally added to or subtracted from the medium. In addition, themedium may further contain an IGF2 inhibitor. As the IGF2 inhibitor,chromeceptin may be typically mentioned.

In a case where light outgrowth cells are selected and induced todifferentiate into chondrocytes according to the method of the presentinvention, not only significantly high expression levels of chondrocytemarkers are observed as compared with a chondrogenic pellet derived fromheavy OG cells, but also a chondrogenic pellet having a histologicallystable structure can be generated. Therefore, in the method in whichonly light cells are selected and induced to differentiate intochondrocytes so that the chondrocytes are produced, according to themethod of the present invention, not only differentiation efficiencywhich induces differentiation of stem cells into chondrocytes can beimproved as compared with a conventional method, but also quality ofdifferentiated chondrocytes can be improved. Thus, such a method can beusefully used in the treatment of a cartilage damage disease inregenerative medicine.

In addition, the present invention provides a chondrocyte produced bythe method of the present invention.

In addition, the present invention provides a pharmaceutical compositionfor preventing or treating a cartilage damage disease, comprising thechondrocyte as an active ingredient.

In the pharmaceutical composition of the present invention, thecartilage damage disease may be preferably at least one selected fromthe group consisting of degenerative arthritis, rheumatoid arthritis,fracture, plantar fasciitis, humerus epicondylitis, calcified myositis,nonunion of fracture, or joint injury caused by trauma. However, thepresent invention is not limited thereto, and any disease known in theart as a disease of cartilage area which may be caused by a cartilagedefect or damage can be included without limitation.

A therapeutically effective amount of the composition of the presentinvention may vary depending on a variety of factors, such as method ofadministration, target site, and the patient's condition. Therefore, ina case of being used in the human body, the dosage should be determinedto an appropriate amount, taking into consideration together with safetyand effectiveness. It is also possible to estimate an amount to be usedin humans from the effective amount determined through animalexperiments. Such considerations in determining the effective amount aredescribed, for example, in Hardman and Limbird, eds., Goodman andGilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001),Pergamon Press; and E. W. Martin ed., Remington's PharmaceuticalSciences, 18th ed. (1990), Mack Publishing Co.

The composition of the present invention may also contain carriers,diluents, excipients, or a combination of two or more thereof, commonlyused in biological preparations. Pharmaceutically acceptable carriersare not particularly limited as long as they are suitable for in vivodelivery of the composition. As such pharmaceutically acceptablecarriers, for example, compounds described in Merck Index, 13th ed.,Merck & Co. Inc., saline, sterile water, Ringer's solution, bufferedsaline, dextrose solution, maltodextrin solution, glycerol, ethanol, anda mixture of one or more thereof can be used, and if necessary, othertypical additives such as antioxidants, buffers, and bacteriostaticagents can be added thereto. Furthermore, the composition can bepreferably made into preparations, depending on respective diseases oringredients, using appropriate methods in the art or methods disclosedin Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

Hereinafter, the present invention will be described in more detail byway of examples.

It will be apparent to those skilled in the art that these examples aregiven to merely illustrate the present invention and that the scope ofthe present invention is not construed as being limited by theseexamples.

Experimental Methods

Hereinafter, specific experimental methods performed for the examples ofthe present invention will be described. The following experimentalmethods indicate some methods of carrying out the examples of thepresent invention and may be optionally changed by a person havingordinary skill in the art.

1) Isolation of CBMCs

CBMCs were acquired from the Cord Blood Bank at Seoul ST. Mary'sHospital. Cord blood was diluted with phosphate buffered saline (PBS)and centrifuged at 850×g for 30 minutes through a Ficoll gradient. CBMCswere collected, washed, and frozen. The frozen CBMCs were thawed andresuspended in StemSpan medium (STEMCELL Technological, Vancouver,British Columbia, Canada) supplemented with CC110 cytokine cocktail(STEMCELL) before use. Before reprogramming, the cells were kept for 5days at 37° C. in 5% CO₂.

2) Blood Samples and Ethics Regulations

The present study was approved by the Institutional Review Board (IRB)of the Catholic University of Korea.

3) Reprogramming Using Sendai Virus

Reprogramming refers to a process by which epigenetic marks are changedin the course of mammalian development. In general, reprogramming ofinduced pluripotent stem cells refers to a technique of inducingpluripotent stem cells by artificially overexpressing factors necessaryfor reprogramming in somatic cells. The method for overexpressing genesincludes using viruses, plasmid vectors, mRNAs, proteins, or the like.

CBMCs were seeded onto a 24-well plate at a concentration of 3×10⁵.Reprogramming was induced using the CytoTune-iPS Sendai Reprogrammingkit. Infection was performed with a multiplicity of infection of 7.5 per3×10⁵ cell infectious unit. After addition of viral components, thecells were centrifuged for 30 minutes at a condition of 1,160×g and 35°C., and then incubated at 37° C. in 5% CO₂. Next day, the cells weretransferred to a 12-well plate coated with vitronectin (LifeTechnologies) and precipitated by performing centrifugation for 10minutes at 1,160×g and 35° C. After the centrifugation, TeSR-E8 medium(STEMCELL) was added thereto at a ratio of 1:1. The reprogrammed cellswere kept and expanded in TeSR-E8 medium with daily medium replacement.

4) Staining with Alkaline Phosphatase

In order to obtain colonies large enough to be stained, the cells wereinoculated, at a concentration of 2×10³, into a 6-well plate coated withvitronectin, and expanded for 5 to 7 days. Staining of undifferentiatediPSC colonies was performed using an alkaline phosphatase detection kit(Millipore, Billerica, MA, USA). The cells were washed with PBScontaining 0.05% Tween-20 and fixed with 4% paraformaldehyde for 2minutes. Fast Red Violet, Naphthol AS-BI phosphate solution, and waterwere mixed at a ratio of 2:1:1 to prepare a staining reagent. The cellswere washed twice with PB ST. Treatment with the staining solutionmixture was performed at room temperature (RT) for 15 minutes. Afterincubation, the cells were washed with PBST and covered with PBS toprevent drying. The stained colonies were measured with a microscope.

5) Immunocytochemical Staining

In order to obtain iPSC colonies large enough to be stained, the cellswere inoculated, at a concentration of 2×10³, into a 6-well plate coatedwith vitronectin. In order to induce iPSC colonies, the cells wereexpanded for 5 to 7 days with daily medium replacement. After theexpansion, the iPSCs were washed with PBS and fixed with 4%paraformaldehyde. The cells were permeabilized with 0.1% Triton X-100(BIOSESANG) for 10 minutes. After the infiltration, the cells wereblocked with PBS (PBA) containing 2% bovine serum albumin (BSA; SigmaAldrich, St. Louis, Mo., USA) for 30 minutes at room temperature.Primary antibodies were diluted in PBA in the following dilutionproportions: OCT4 (1/100; Santa Cruz, CA, USA), KLF4 (1/250; Abcam,Cambridge, UK), SOX2 (1/100; BioLegend, San Diego, CA, USA), TRA-1-60(1/100; Millipore), TRA-1-81 (1/100; Millipore), and SSEA4 (1/200;Millipore). Incubation with the primary antibodies was performed at roomtemperature for 2 hours. Alexa Fluor 594-(1/400; Life Technologies) and488-(1/400; Life Technologies) conjugated secondary antibodies werediluted with PBA and incubation therewith was performed for 1 hour atroom temperature while avoiding light. The cells were washed and mountedusing ProLong Antifade mounting reagent (Thermo Fisher Scientific,Waltham, Mass., USA). The stained colonies were detected with animmunofluorescence microscope.

6) Polymerase Chain Reaction Using CBMC-iPSC Sample

5×10⁵ iPSCs were harvested and frozen at −20° C. Total mRNA wasextracted therefrom using Trizol (Life Technologies) and cDNA wassynthesized using RevertAid™ First Strand cDNA Synthesis Kit (ThermoFisher Scientific). The synthesized cDNA was used to perform reversetranscriptase polymerization. Primer sequences are shown in [Table 1]below.

TABLE 1 Primer sequences for translocation markers usedin real-time RT-PCR Target Name Direction Primer Sequence Size OCT3/4Forward ACCCCTGGTGCCGTGAA 190 Reverse GGCTGAATACCTTCCCAAATA SOX2 ForwardCAGCGCATGGACAGTTAC 321 Reverse GGAGTGGGAGGAAGAGGT NANOG ForwardAAAGGCAAACAACCCACT 270 Reverse GCTATTCTTCGGCCAGTT LIN28 ForwardGTTCGGCTTCCTGTCCAT 122 Reverse CTGCCTCACCCTCCTTCA DPPB5 ForwardCGGCTGCTGAAAGCCATTTT 215 Reverse AGTTTGAGCATCCCTCGCTC TDGP1 ForwardTCCTTCTACGGACGGAACTG 140 Reverse AGAAATGCCTGAGGAAAGCA GAPDH ForwardGAATGGGCAGCCGTTAGGAA 414 Reverse GACTCCACGACGTACTCAGC

7) Karyotyping

Cells were cultured until confluency reached about 80%. A chromosomeresolution additive (Genial Genetic Solutions, Runcorn, UK) was added toeach well. After incubation, treatment with Colcemid® was performed for30 minutes. The cells were harvested and treated with a preheated stocksolution to become a solution. The resultant was fixed with a mixtureobtained by mixing acetic acid and a methanol solution at a ratio of1:3. Slides were prepared for chromosome analysis using thetrypsin-Giemsa banding technique.

8) Functional Identification of iPSCs

A kit for identifying human pluripotent stem cell function (R&D,Minneapolis, MN, USA) was purchased to evaluate differentiationcapacities of three germ layers. The day before the experiment, aculture dish was coated with Cultrex PathClear BME (R&D) according tothe manufacturer's instructions. A medium specific to each germ layerwas prepared and cells were cultured individually. Afterdifferentiation, the cells were washed with PBS and fixed with 4%paraformaldehyde. Permeation and blocking were performed with 0.3%Triton X-100 and 1% PBA for 45 minutes. Antibodies against Otx2 (1/10,ectoderm), Brachyury (1/10 mesoderm), and Sox17 (1/10, endoderm) werediluted. The antibodies were suspended in PBA and incubation therewithwas performed at room temperature for 3 hours.

After washing the primary antibodies, Alexa Fluor 568 donkey anti-goatsecondary antibodies (1:200; R&D) were diluted with PBA and incubationtherewith was performed for 1 hour. The cells were washed with PBA, andtreatment with a DAPI solution was performed at room temperature for 10minutes. The cells were washed and covered with PBS. The stainingresults were checked using a fluorescence microscope.

9) EB-Derived Outgrowth Cell Induction

CBMC-hiPSCs were expanded and 2×10⁶ cells were prepared. The cells wereresuspended in Aggrewell medium (STEMCELL) and seeded on a 100-mmculture dish. The cells were cultured for one day at 37° C. in 5% CO₂.Next day, the medium was replaced with TeSR-E8 medium and the cells werekept expanded for 6 days. After the expansion process, EBs wereharvested and resuspended in DMEM containing 20% fetal bovine albumin(FBS). The resultant was placed on a gelatin-coated dish to induceoutgrowth cells. The cells were kept for one week at 37° C. in 5% CO₂prior to chondrogenic differentiation.

10) Chondrogenic Differentiation Using EB-Derived Outgrowth Cells

The outgrowth cells derived from the EBs were washed and separated fromthe culture dish. The cells were passed through a 40 μm cell strainer(Thermo Fisher Scientific) to remove cell masses. Single outgrowth cellswere counted and 3×10⁵ cells per chondrogenic pellet were prepared.3×10⁵ outgrowth cells were cultured in a chondrogenic differentiationmedium (DMEM, 20% knockout serum replacement, 1× non-essential aminoacids, 1 mM L-glutamine, 1% sodium pyruvate, 1% ITS+Premix, 10⁻⁷ Mdexamethasone, 50 μm ascorbic acid, 40 μg/mL of L-proline, supplementedwith 50 ng/mL of human bone morphogenetic protein 2, and 10 ng/mL ofhuman transforming growth factor beta 3), and transferred to a conicaltube. The cells were centrifuged at 750×g for 5 minutes. The resultingchondrogenic pellet was kept for 30 days and replacement of the culturemedium was performed daily. BMSCs were used as a positive control.

11) Histological Analysis of Chondrogenic Pellet

The chondrogenic pellet was fixed with 4% paraformaldehyde at roomtemperature for 2 hours. One layer of gauze was placed on a cassette andthe pellet was transferred to the gauze. Dehydration was performedsequentially with an ethanol solution. The dehydration solution wasremoved with a mixture of graded ethanol and zylene (Duksan PureChemical Co., Ltd., Ansan, Korea) and paraffin infiltration wasperformed overnight. Next day, the pellet was immobilized on a paraffinblock and a 7 μm section was obtained using a microtome. The slide wasdried at 60° C. for 2 hours. The section was deparaffinized with 2cycles of zylene. The section was rehydrated with decreasing sequentialethanol series and washed with tap water for 5 minutes.

For alcian blue staining, the section was incubated in 1% alcian bluesolution for 30 minutes. Then, the slide was washed and counter-stainedwith nuclear fast red for 1 minute. Safranin O staining was performed byincubating the slide in Weigert's iron hematoxylin for 10 minutes. Slidewas washed and incubated in 0.1% safranin O solution for 5 minutes.

For toluidine staining, the section was incubated in toluidine bluesolution for 4 minutes. After the staining process, the section waswashed and passed through increasing sequential ethanol series. Ethanolwas removed with 2 cycles of zylene and the slide was fixed usingVectaMount™ Permanent Mounting Medium (VectorLaboratories). Staining waschecked with a microscope.

12) Immunohistochemistry

The section was dried at 60° C. for 2 hours and deparaffinized with 2cycles of zylene. The section was rehydrated with decreasing sequentialethanol series and washed with tap water for 5 minutes. Antigenunmasking was induced by incubation in citrate buffer for 15 minutes andcooling for 20 minutes. The cooled section was washed twice withdeionized water (DW). Activity of endogenous peroxidase was blocked byincubating the section in 3% hydrogen peroxide diluted in DW for 10minutes. The section was washed twice with DW and then further washedwith Tris buffered saline (TBS) containing 0.1% Tween-20 (TBST). Thesection was blocked with TBS containing 1% BSA at room temperature for20 minutes. Primary antibodies diluted in blocking solution were addedto the section and incubation was performed overnight at 4° C. Theprimary antibodies were diluted in the following proportions: type 1collagen (1/100, Abcam), type 2 collagen (1/100, Abcam), and aggrecan(1/100, GeneTex, Irvine, CA, USA). A negative control slide was treatedwith the same amount of blocking solution containing no antibody. Nextday, the section was washed three times for 3 minutes each in TBST, andincubation with secondary antibodies (1/200) was performed at roomtemperature for 40 minutes. The section was washed with TBST and ABCreagent and incubated for 30 minutes. The slide was washed 3 times withTBST and a DAB solution (Vector Laboratories) was applied for 1 minute.The section was washed with DW until the color was washed away. Mayer'shematoxylin was applied to the section for 1 minute for counterstaining. The section was washed and passed through increasingsequential ethanol series. Ethanol was removed with two cycles of zyleneand the slide was mounted using VectaMount™ Permanent Mounting Medium(Vector Laboratories). Staining was checked with a bright-fieldmicroscope.

13) Polymerase Reaction of Chondrogenic Pellet

chondrogenic pellets were harvested at each time point and frozen at−80° C. The samples were rapidly frozen with liquid nitrogen and groundwith pestle and mortar. Each of the ground pellet samples was incubatedwith Trizol for mRNA extraction. cDNA was synthesized from the extractedmRNA, and polymerase chain reaction was performed with primers forcell-specific markers. Primer sequences for RT-PCR are shown in [Table2]. Primer sequences for real-time PCR are shown in [Table 3] below. Themean cycle threshold obtained from triplicate experiments was used tocalculate gene expression so as to average GAPDH as an internal control.

TABLE 2 Primer sequences used in RT-PCR for amplificationof chondrogenic markers Target Name Direction Primer Sequence Size SOX9Forward GAACGCACATCAAGACGGA 631 G Reverse TCTCGTTGATTTCGCTGCTC ACANForward TGAGGAGGGCTGGAACAA 349 GTACC Reverse GAGGTGGTAATTGCAGGGA ACACOL2A1 Forward TTCAGCTATGGAGATGACA 472 ATC Reverse AGAGTCCTAGAGTGACTGA GCOMP Forward CAACTGTCCCCAGAAGAGC 588 AA Reverse TGGTAGCCAAAGATGAAGC CCCOL1A1 Forward CCCCTGGAAAGAATGGAGA 148 TG Reverse TCCAAACCACTGAAACCTC TGCOL10 Forward CAGTCATGCCTGAGGGTTT 196 T Reverse GGGTCATAATGCTGTTGCC TGAPDH Forward GAATGGGCAGCCGTTAGGA 414 A Reverse GACTCCACGACGTACTCAG C

TABLE 3 Primer sequences used in real-time PCR foramplification of chondrogenic marker Target Name DirectionPrimer Sequence Size SOX9 Forward TTCCGCGACGTGGACAT  77 ReverseTCAAACTCGTTGACATCGAA GGT ACAN Forward AGCCTGCGCTCCAATGACT 107 ReverseTAATGGAACACGATGCCTTT CA COL2A1 Forward GGCAATAGCAGGTTCACGT  79 ACARverse CGATAACAGTCTTGCCCCAC TTA COMP Forward AGCAGATGGAGCAAACGTA  76 TTGReverse ACAGCCTTGAGTTGGATGCC COL1A1 Forward CCCCTGGAAAGAATGGAGA 148 TGReverse TCCAAACCACTGAAACCTCT G COL10 Forward CAGTCATGCCTGAGGGTTTT 196Reverse GGGTCATAATGCTGTTGCCT

[Example 1] Production of hiPSCs Using Isolated CBMCs

Reprogramming of CBMCs was facilitated using sendai virus containing aYamanaka factor. The Yamanaka factor is a gene capable of inducingpluripotency. Some time after transduction, CBMC-hiPSCs formed a colonysimilar to embryonic stem cells (FIG. 1A). The CBMC-hiPSCs were purifiedinto a cell line with the same cell morphology. The same CBMC-hiPSCswere used for further characterization. The established CBMC-hiPSCs werestained with alkaline phosphate (FIG. 1B). Expression of pluripotencymakers including OCT4, SOX2, NANOG, LIN28, KLF4, and c-MYC was alsomeasured (FIG. 1C). The parental CBMCs were used as a negative control.Expression of OCT4, SOX2, NANOG, and LIN28 was increased in theCBMC-hiPSCs. However, low expression of KLF4 and c-MYC was observed inthe CBMC-hiPSCs as compared with the CBMCs. Typical cell surface markers(SSEA4, OCT4, SOX2, KLF4, TRA-1-80, and TRA-1-60) were identified byimmunochemical analysis (FIG. 1D). All differentiated cell linesexpressed markers which become standards for pluripotency. It wasidentified that the CBMC-hiPSCs maintain their normal karyotype evenafter the reprogramming process and also differentiate into various germcells. These data indicate that CBMC-hiPSCs have been successfullyproduced and have pluripotency.

[Example 2] Differentiation of CBMC-iPSCs into Chondrocytes

In order to check cartilage regeneration capacity of CBMC-iPSCs,chondrogenic differentiation was performed through culture of EBs andinduction of outgrowth cells. A simple scheme for a chondrogenic pelletproduction process is as illustrated in FIG. 2A. CBMC-iPSCs colonieswere prepared for chondrogenic differentiation (FIG. 2B). The CBMC-iPSCswere expanded and aggregated into EBs (FIG. 2C). The EBs were expandedfor several days and transferred to a gelatin-coated culture dish so asto be induced to outgrowth cells (FIG. 2D). The outgrowth cells wereexpanded and were isolated into single cells for differentiation intochondrocytes. 2×10⁶ iPSCs were used to obtain a number of chondrogenicpellets. After 30 days of differentiation, the chondrogenic pellets weregenerated using the EB outgrowth cells. The generated chondrogenicpellets exhibited a three-dimensional spheroidal shape. From the above,it was identified that CBMC-hiPSCs can differentiate into chondrocytesand can form a cartilage shape having a spheroidal shape due toaccumulation of ECM.

[Example 3] Identification of Expression of Cartilage Gene inChondrogenic Pellet

Through previous procedures, chondrogenic pellets were successfullygenerated from CBMC-hiPSCs. In addition, the differentiated cellssynthesized ECM components and exhibited cartilage-like characteristics.Expression of major ECM constitutive proteins such as aggrecan (ACAN),type 2 collagen (COL2A1), and cartilage oligomeric matrix protein (COMP)was respectively checked at day 10, day 20, and day 30. As a result, itwas identified that expression of ACAN, COL2A1, and COMP is increased(FIG. 3 ). Sex-determining region Y-box 9 (Sox9) is known as atranscription factor that regulates gene expression of earlychondrogenic differentiation markers and ECM proteins. Expression ofSox9 was increased after 20 days. That is, genetic characteristics ofthe generated chondrogenic pellets were identified. Corresponding tocartilage-like morphology, an increase in gene expression of major ECMcomponent proteins was identified.

[Example 4] Histological Characteristics of Chondrogenic Pellet

As increased expression of chondrogenic markers was identified, proteinlevels in the chondrogenic pellets generated from CBMC-hiPSCs wereevaluated by histological analysis (FIG. 4 ). Safranin O staining,alcian blue staining, and toluidine blue staining are staining methodsused for detection of ECM in cartilage. As a result of the abovestainings, accumulation of ECM was identified in the inner part of thepellet even at an early stage of differentiation (day 10). Lacuna is oneof the major features appearing in articular cartilage. A reservoir likeempty lacuna was seen after 10 days. However, the size thereof wasdecreased as differentiation progressed. At day 30 of differentiation,the reservoir seemed like lacuna as ECM was accumulated in thereservoir. The staining intensity at day 30 was almost similar.

The quality of cartilage is determined by the major types of ECMproteins. Therefore, it is important to identify specific proteins.Aggrecan and type 2 collagen proteins are known as main components thatconstitute ECM. Type 2 collagen is a major collagen type that representsvitreous cartilage. Antibodies against type 2 collagen and aggrecan werestained against the chondrogenic pellet for chondrogenic differentiation(FIG. The staining intensity of type 2 collagen was higher in theCBMC-hiPSC-derived chondrogenic pellet than the MSC control.Corresponding to the previous staining results, aggrecan and type 2collagen were mostly detected in the inner part of the pellet at day 30.A major feature of fibrous cartilage is high expression of type 1collagen. It was identified that the chondrogenic pellet does not havethe predominant feature of fibrous cartilage (FIG. Expression of type 1collagen was relatively higher than that in MSC control mice. However,expression remained at a certain level and did not remarkably increaseduring differentiation. The chondrogenic pellet generated fromCBMC-hiPSCs is characterized by having a similar quality to thechondrogenic pellet derived from MSCs after 30 days of differentiation.Chondrocytes differentiated from CBMC-hiPSCs were capable of producingECM component proteins. Type 2 collagen was expressed at a higher levelthan type 1 collagen in the CBMC-hiPSC-derived chondrogenic pellets. Inconclusion, it was identified that CBMC-hiPSCs can producecartilage-like features similar to those of vitreous cartilage.

[Example 5] Analysis for Genetic Markers in Chondrogenic Pellets Derivedfrom CBMC-hiPSCs and MSCs

Collagen is the most abundant protein that constitutes ECM. There aremany types of collagen, but collagen types 1, 2, and 10 are mainlyrelated to cartilage. In previous experiments, expression of type 1collagen and type 2 collagen was identified by histological analysis(FIGS. 5A and 5B). Based on the above results, expression of type 1collagen (COL1A1) gene was analyzed (FIG. 6A). Gene expression of type10 collagen (COL10), a protein known to be dominant type expressed inhypertrophic cartilage, was also analyzed. Stable expression of type 1collagen was identified by histochemical staining. However, expressionof COL1A1 decreased at each time point. Expression of COL10 did notchange during the differentiation process. As mentioned earlier, theproportion of type 2 collagen can alter the resulting characteristics ofthe chondrogenic pellet. Using the previous gene expression data, thegene expression ratio of COL2A1 to COL1A1 was evaluated (FIG. 6B). Thetotal increase rate indicates that the hyaline cartilage gene is highlyexpressed relative to the fibrous cartilage gene. The CBMC-hiPSC-derivedchondrogenic pellet was compared with the chondrogenic pellet generatedin BMSCs at day 30 using real-time PCR. There was no statisticalsignificance of ACAN expression between the two samples. Expression ofCOL2A1 and SOX9 was significantly higher in the chondrogenic pelletdifferentiated from CBMC-hiPSCs than in the BMSC-derived chondrogenicpellet. However, COMP was highly expressed in the MSC controlchondrogenic pellet. Expression of the fibrous marker COL1A1 was alsohigher in the MSC control chondrogenic pellet. However, expression ofthe hypertrophy marker COL10 was remarkably lower in theCBMC-hiPSC-derived chondrogenic pellet. These results highlightpossibility of CBMC-hiPSCs as a potential cell source for cartilageregeneration in future applications.

[Example 6] Identification of Differentiation Capacity Increasing Effectof Chondrocytes Differentiated from CBMC-Derived iPSCs

In order to identify that differentiation capacity can be improved inthe method for differentiation into chondrocytes of the presentinvention, the present inventors induced differentiation intochondrocytes using peripheral blood cell (PBMC)-derived iPSCs andCBMC-derived iPSCs of the present invention. After differentiation, therespective chondrocytes were obtained, from which expression levels ofACAN and COL2A1 associated with cartilage formation were checked.

As a result, as illustrated in FIG. 7 , it was identified thatexpression levels of ACAN and AOL2A1 are about 6 to 10 times higher inthe CBMC-derived chondrocytes than those of the PMBC-derivedchondrocytes. From this, it was identified that cartilage productionefficiency is increased in the CBMC-derived chondrocytes of the presentinvention.

[Example 7] Identification of Whether Prepared iPSCs are Homozygotes

In order to identify whether the prepared CMC-hiPSCs are homozygotes,allele types of Human Leukocyte Antigen (HLA) were analyzed for threeCMC-derived iPSC cell lines. As a result, as shown in [Table 4] to[Table 6], it was identified that the CMC-hiPSCs prepared by the methodof the present invention are homozygotes.

Accordingly, the chondrocytes differentiated using the CMC-iPSCs of thepresent invention can be used in the form of chondro beads to producecartilage tissue, and an increased transplantation success rate can beexhibited.

TABLE 4 Results of HLA test (SBT) HLA-A HLA-B HLA-C DRB1 DQB1 DPB1CMC-hiPSC- *33:03(A33) *44:03(B44) — *13:02 — — 008 *33:03(A33)*44:03(B44) — *13:02 — —

TABLE 5 Results of HLA test (SBT) HLA-A HLA-B HLA-C DRB1 DQB1 DPB1CMC-hiPSC- *24:02(A24) *07:02(B7) — *01:01 — — 009 *24:02(A24)*07:02(B7) — *01:01 — —

TABLE 6 Results of HLA test (SBT) HLA-A HLA-B HLA-C DRB1 DQB1 DPB1CMC-hiPSC- *11:01(A11) *15:01(B62) — *04:06 — — 008 *11:01(A11)*15:01(B62) — *04:06 — —

For production of iPSCs, the homozygous type that can account for thehighest proportion of the Korean population was selected. However, HLAtypes of Koreans are treated as confidential due to the PersonalInformation Protection Law. Accordingly, in the present study, HLA typeinformation of CBMCs which had been legally donated and were stored inthe Catholic Hematopoietic Stem Cell Bank was analyzed. As shown in[Table 7] below, information on the top 20 HLA types was obtained.HLA-A*33, HLA-B*44, and HLA-DRB1*13 accounted for approximately 23.97%in the entire CBMC bank, and thus were the most frequent homozygous HLAtypes. The second most frequent types were HLA-A*33, HLA-B*58, andHLA-DRB1*13 which were recorded as accounting for 11.16% of theestimated population. In the top 5 types, the coverage of HLA types wasless than 5%. Among the three HLA types, HLA-A tended to be relativelymore concentrated than the other two types. Of the 20 selected HLAtypes, 25% had HLA-A type of *33 and 40% had HLA-A type of *02.

Based on the information of the classified HLA types, 9 homozygous cellswere selected for recombination in the Catholic Hematopoietic Stem CellBank. In addition, 4 PBMC samples with homozygous HLA types wereobtained as a gift. As shown in [Table 8] below, 3 of the total 13 cellsamples had HLA types of HLA-A*33, HLA-B*44, and HLA-DRB1*13. Theobtained cells were transferred to the GMP facility at the CatholicInstitute of Cell Therapy for reprogramming. After treatment with aYamanaka factor, samples with no defect were isolated and characterizedby various assays. Survival was measured based on cell attachment.Pluripotency was identified through PCR and immunofluorescence.Undifferentiated state of the cells was identified by alkalinephosphatase staining. Karyotyping and short tandem repeat assay wereperformed to identify the normal genetic background. Finally, multipletransfers thereof were identified through differentiation intorespective germ layers, and infection testing was performed. Afterundergoing these processes, only cells that passed all quality controltests were stored in the GMP facility (FIG. 8A).

After several subcloning procedures, all cell lines showed a proper formof iPSCs. Homozygous HLA-iPSCs showed positive expression ofpluripotency markers (FIGS. 8B and 8D). All cells remainedundifferentiated during passaging (FIG. 8C). Normal karyotype wasidentified in the cell line (FIG. 8E). In addition, the produced cellswere capable of differentiating into all three germ layers in vitro(FIGS. 8F and 8G). The data illustrated in FIGS. 8B-8F represents datafor the CMC-hiPSC-001 cell line.

Production of homozygous HLA-iPSCs opened up new opportunities fordevelopment of personalized regenerative medicine. By reducing the time,money and manpower required, the homozygous HLA-iPSCs can be used totreat a large number of patients with minimal cell sources. Depending onthe HLA phenotype allele frequency, candidate HLA homozygous cell typescan be selected and held as an iPSC resource bank. However, homozygouscells are not frequently found, and thus it is important to estimate theminimum or appropriate number of iPSCs that accounts for the largestpercentage of the population.

As a result, the present inventors determined the frequencies ofhomozygous HLA types in the Korean population with an alternativemethod. The present inventors firstly accessed the HLA-typed CBMClibrary of the Catholic Institute of Cell Therapy. Through the CBMCbank, it was identified that 23.9% of the HLA-homozygous CBMCs stored atthe bank show phenotypes of HLA-A*33, HLA-B*44, and HLA-DRB1*13.Homozygous single cells were screened through the produced data andreprogrammed so that the cells are obtained as iPSCs. The threehomozygous HLA-iPSCs, HLA-A*33, HLA-B*44, and HLA-DRB1*13 were produced.13 produced HLA-iPSC lines had high pluripotency and normal karyotypeand passed various contamination tests. This is the first achievement inwhich the Korean homozygous HLA-iPSC bank has been established inKoreans under the sponsorship of the Korean government. HomozygousHLA-iPSCs will open up new opportunities for successful regenerativemedicine and clinical stem cell therapy.

TABLE 7 HLA-A HLA-B HLA-DR(B1) % of Frequency 1 *33 *44 *13 23.97 2 *33*58 *13 11.16 3 *24 *07 *01 7.85 4 *30 *13 *07 7.44 5 *33 *44 *07 7.02 6*24 *52 *15 4.55 7 *11 *15 (62) *04 4.13 8 *24 *54 *04 2.48 9 *02 *46*08 2.07 10 *01 *37 *10 1.65 11 *02 *27 *01 1.65 12 *02 *15 (62) *041.24 13 *24 *51 *09 1.24 14 *33 *58 *03 1.24 15 *33 *58 *15 1.24 16 *02*13 *12 0.83 17 *02 *46 *09 0.83 18 *02 *48 *04 0.83 19 *02 *48 *14 0.8320 *02 *51 *15 0.83

TABLE 8 HLA Type Donor No. Cell Type A B DR(B1) CMC-hiPSC-001 PBMC*33:03 *44:03 *13:02 CMC-hiPSC-002 PBMC *33:03 *44:03 *07:01CMC-hiPSC-003 PBMC *33:03 *44:03 *13:02 CMC-hiPSC-004 PBMC *33:03 *44:03*07:01 CMC-hiPSC-005 CBMC *33:03 *58:01 *13:02 CMC-hiPSC-006 CBMC *33:03*44:03 *07:01 CMC-hiPSC-007 CBMC *02:01 *48:01 *14:54 CMC-hiPSC-008 CBMC*33:03 *44:03 *13:02 CMC-hiPSC-009 CBMC *24:02 *07:02 *01:01CMC-hiPSC-010 CBMC *02:01 *51:01 *04:03 CMC-hiPSC-011 CBMC *11:01 *15:01*04:06 CMC-hiPSC-012 CBMC *33:03 *58:01 *13:02 CMC-hiPSC-013 CBMC *33:03*58:01 *13:02

[Example 8] Construction of Minicircle Vector Encoding Human GrowthFactor

<8-1> Construction of Minicircle Vector Expressing BMP2 or TGFβ3 Inorder to induce expression of BMP2 and TGFβ3 in the present invention,minicircle vectors were constructed in the same procedure as illustratedin FIG. 9A.

Specifically, for human BMP2 and TGFβ3 genes, cDNA sequences weresynthesized as the base sequences of SEQ ID NO: 1 and SEQ ID NO: 2,respectively, by optimizing codons and used. The synthesized cDNAsequence was inserted into a parental plasmid(CMV-MCS-EF1-RFP-SV40-PolyA; manufacturer: System Biosciences, MountainView, CA, USA) as a mock vector. At the time of insertion, the BMP2and/or TGFβ3 sequences were inserted into the sequence between BamHI andXbaI in a multiple cloning site downstream of the CMV promoter, toconstruct a parental vector containing the growth factor gene. Eachparental vector (ppBMP2, pp TGFβ3) containing BMP2 or TGFβ3 wasrespectively transformed into ZYCY10P3S2T E. coli cells. The transformedcells were isolated into single unit colonies, which were inoculated in2 ml of LB medium containing 500 μl/ml of kanamycin and initiallycultured at 30° C. for 2 hours. Then, 200 ml of terrific broth (TB) wasadded to a 1 L culture flask, 100 μl of the initially cultured mediumwas inoculated thereinto, and the culture flask was cultured for 15hours with shaking culture at 30° C. with 200 rpm. After the culture,200 ml of LB medium containing 200 μl of 4% 1N NaOH and 20% L-arabinosewas added to the culture flask so as to convert the parental vectorplasmid into a minicircle vector. The flask to which the medium had beenadded was further cultured at 30° C. with 200 rpm for 5 hours. Aftercompletion of the culture, the cells were obtained and plasmid DNA wasextracted using the NucleoBond Xtra plasmid purification kit(Macherey-Nagel, Duren, Germany). The extracted DNA was doubly cleavedwith XbaI and BamHI to check the size of the constructed minicircle andthe inserted BMP2 or TGFβ3 gene. The respective vectors into which BMP2and TGFβ3 had been inserted were named mcBMP2 and mcTGFβ3, respectively.In order to prepare a negative control, a minicircle vector (mcMock) wasconstructed in the same manner using a parental plasmid (ppMock) intowhich BMP2 and TGFβ3 had not been inserted.

As a result, as illustrated in FIG. 9B, it was identified that the sizeof mcBMP2 is about 7.3 kb and appears as two bands of about 1.1 kb ofBMP2 gene and about 5 kb of minicircle in a case of being doubly cleavedwith restriction enzymes. In addition, it was identified that the sizeof mc TGFβ3 is about 7.5 kb and appears as two bands of about 1.3 kb ofTGFβ3 gene and about 5 kb of minicircle in a case of being doublycleaved with restriction enzymes.

<8-2> Identification of Transduction Efficiency of mcBMP2 and mcTGFβ3

In order to identify whether mcBMP2 and mcTGFβ3 are capable ofsignificantly exhibiting expression activity in a case of beingtransduced into cells, expression levels of RFP which is also present inboth mcBMP2 and mcTGFβ3 were measured.

Specifically, mcBMP2 or mcTGFβ3 constructed in Example <8-1> wasrespectively mixed with lipofectamine in Opti-MEM medium (Thermo FisherScientific) for 20 minutes. Then, the mixed DNA-lipofectamine mixturewas added to HEK293T cell culture medium and incubated for 6 hours in a5% CO₂ incubator at 37° C. After the incubation, the cells were checkedfor cell morphology with a phase contrast microscope, and thenexpression levels of RFP in HEK293T cells were observed using afluorescence microscope. In addition, a level of protein expressed inHEK293T cells transformed with mcBMP2 or mcTGFβ3 was identified bychecking a level of protein expression with the Bradford assay for themedium in which the cells had been cultured. For quantitative analysis,absorbance values were relatively compared on the basis that theabsorbance value of the recombinant BMP2 protein (rhBMP2) or recombinantTGFβ3 protein solution is 1.0.

As a result, as illustrated in FIGS. 10A-10D, it was identified that thehighest expression level of RFP is observed in mcMock-transduced cellsas compared with HEK293T cells transduced with mcBMP2 and mcTGFβ3,indicating that mcMock exhibits the highest transduction efficiency(FIGS. 10A and 10B). In addition, it was identified that in a case wherea level of protein expressed and secreted in the cell culture medium ischecked, the supernatant of the HEK293T cells transformed with mcBMP2exhibits a significantly high level of absorbance, indicating anexpression level of about 0.1 mg/ml (FIG. 10C). As compared with this,it was identified that expression of mcTGFβ3 is observed at a relativelylow level. However, as compared with the culture supernatant of mcMock,it was identified that the cells are capable of exhibiting a significantprotein expression level as transduced with mcTGFβ3 (FIG. 10D).

[Example 9] Induction of Differentiation into Human iPSC-DerivedChondrocytes Using mcBMP2 and mcTGFβ3

A method of differentiating human-derived iPSCs (hiPSCs) intochondrocytes was performed according to the procedure of the schematicdiagram illustrated in FIG. 11A. All experiments were repeated threetimes in total for the same experimental group.

i) Step of preparing iPSCs The method for obtaining iPSCs from cordblood mononuclear cells (PBMCs) was carried out with a reprogrammingmethod according to the conventional dedifferentiation induction method[NPL 1]. The obtained iPSCs were cultured in a container coated withvitronectin (Thermo Fisher Scientific, Waltham, Mass., USA), and theculture was performed using E8 medium (STEMCELL Technologies) as aculture medium with daily medium replacement. The morphology of theprepared iPSCs is as illustrated in FIG. 11B.

ii) Step of generating embryoid bodies (EBs) from iPSCs: The preparediPSCs were detached from the bottom of the container. The detached iPSCswere counted to 2×10⁶ cells and inoculated onto a new plate. A 1:1mixture of TeSR-E8 medium and Aggrewell medium (STEMCELL Technologies)was used as a culture medium. The IPSC cells inoculated into the mixedmedium were cultured in a 5% CO₂ incubator at 37° C. for 24 hours. Then,the medium was removed and replaced with fresh E8 medium. Culture wasperformed for 3 days. Then, the medium was replaced with E7 medium andculture was further performed for 3 days to obtain embryoid bodies (EBs)(FIG. 11C).

iii) Step of inducing EBs to outgrowth cells (OG cells): Then, the EBswere transferred to a gelatin-coated container. For this purpose, theculture container of which the bottom had been coated with 0.1% gelatinfor 30 minutes and completely dried was used. The resulting EBs wereobtained and suspended in an OG induction medium. As the OG inductionmedium, DMEM (Thermo Fisher Scientific) medium containing 20% fetalbovine serum (FBS, Thermo Fisher Scientific) and 10%penicillin/streptomycin (Thermo Fisher Scientific) was used. The EBswere inoculated into a gelatin-coated container at a density of 50 to 70EBs/cm², and cultured in a 5% CO₂ incubator at 37° C. for 3 days so thatprotruding cells like branches (outgrowth cells (OG cells)) werecultured and induced. Morphology of the induced OG cells is asillustrated in FIG. 11D.

iv) Step of transducing OG cells with minicircle vectors: Then, the OGcells were detached and the remaining EB clumps were removed with a 40μm cell strainer (BD Technologies, Franklin Lakes, NJ, USA) so that OGcells in single unit cells were obtained (FIG. 11E). The OG cellsrepresent fibrous morphology similar to mesenchymal stem cells. Theobtained OG cells were again inoculated into a new gelatin-coatedcontainer at a density of 1 to 5×10 4 cells/cm 2 and transduction wasperformed with the minicircle vectors constructed in Example <8-1>. Onthe day prior to transduction, the culture medium was replaced with DMEMmedium containing no serum and antibiotics, and culture was performedovernight. Then, transduction was performed with mcMock, mcBMP2, ormcTGFβ3 using Lipofectamine 2000 reagent (Thermo Fisher Scientific). Forthe cells transduced with the minicircle vectors, identification ofwhether transduction had occurred was made by checking the expressionlevel of intracellular RFP using a fluorescence microscope (FIGS. 11F to11H). It was identified that the OG cells transduced with mcMock exhibita high level of RFP expression, and that the OG cells transduced withmcBMP2 or mcTGFβ3 exhibit a low level of RFP expression as compared withmcMock, similar to the expression pattern of RFP in HEK293T cells.

v) Step of inducing differentiation of OG cells transduced withminicircle vectors into chondrogenic pellet: The OG cells for whichtransduction had occurred due to overnight culture were prepared in a 15ml conical tube to give 3×10 5 cells per pellet, and cultured in achondrogenic differentiation medium. The chondrogenic differentiationmedium (CDM) used a composition of DMEM medium which contains 20%knockout serum replacement, 1× non-essential amino acid, 1 mML-glutamine, 1% sodium pyruvate, 1% ITS+Premix, 10⁻⁷ M Dexamethasone, 50mM ascorbic acid, and 40 μg/ml of L-proline. No recombinant growthfactors such as BMP2 and TGFβ3 were added to the CDM. The OG cells weresuspended in the CDM medium, precipitated by centrifugation at 750×g for5 minutes, and then cultured for 30 days to differentiate into achondrogenic pellet. The medium was replaced at intervals of 3 days.After completion of the final culture, the differentiated chondrogenicpellet was obtained. The obtained chondrogenic pellet was kept frozen at−80° C. before use.

[Example 10] Characterization of Cells Produced by DifferentiationInduction Using Minicircle Vectors

<10-1> Characterization of OG Cells Induced from iPSCs

In order to identify characteristics of the OG cells cultured in thestep iii) of inducing EBs to OG cells, expression levels of mesenchymalstem cell (MSC) markers were checked.

The OG cells induced in the step iii) of <Example 8> were obtained andsuspended in Trizol (Thermo Fisher Scientific) so that the cells weredisrupted and mRNA was extracted therefrom. Using the extracted mRNA asa template, cDNA was synthesized with the RevertAid™ First Strand cDNAsynthesis kit (Thermo Fisher Scientific). Using the synthesized cDNAagain as a template, PCR was performed on the MSC marker genes CD44,CD73, CD90, CD105, and CD45, and expression levels of the respectivegenes were checked. Quantitative analysis was performed by repeatedlychecking the expression level three times for the same gene, and thenthe value of each gene expression level was corrected based on theexpression level of GAPDH in the same cells. In addition to the OGcells, an expression level of the same gene was checked for iPSCs.

First, in a case of checking efficiency when the minicircle vectors aretransduced into the OG cells, it was identified that efficiency of theminicircle vectors transduced into the OG cells shows a similar tendencyto that of HEK293T cells (FIG. 12A). In addition, expression of MSCmarker genes was checked. As a result, it was identified that the OGcells exhibit significant expression levels of CD44, CD73, and CD105,which are somewhat low expression levels as compared with MSCs and aresignificantly high expression levels as compared with hiPSCs (FIG. 12B,FIG. 12C and FIG. 12E). On the contrary, it was identified that CD90 wasexpressed at a somewhat higher level in the OG cells than MSCs (FIG.12D). In addition, it was identified that CD45, known to be a markerwhich is negatively expressed in mesenchymal stem cells, is expressed ata low level in both MSCs and the OG cells (FIG. 12F).

In addition, it was intended to identify differentiation potential ofMSCs. MSCs are known to be able to differentiate into the threelineages, adipocytes, chondrocytes, and osteoblasts. The presentinventors performed alizarin red staining, oil red O staining, andalcian blue staining to identify lineage differentiation capacity of theOG cells induced from iPSCs.

As a result, as illustrated in FIGS. 12G to 12I, it was identified thatthe OG cells exhibit pluripotency which makes it possible todifferentiate into chondrocyte lineage (FIG. 12G), to differentiate intoadipocyte lineage (FIG. 12H), and to differentiate into chondrogenicpellet (FIG. 12I).

<10-2> Identification of Expression Efficiency of Intracellular GrowthFactor Protein Produced by Differentiation Induction Using mcBMP2 andmcTGFβ3

In order to identify expression efficiency of the minicircle vectors inthe chondrogenic pellet produced by differentiation induction in thestep v) of <Example 8>, expression of RFP and GFP in the cells which arein a differentiation induction process was checked. OG cells (mcBMP2-OG)into which mcBMP2 had been transduced, OG cells (mcTGFβ3-OG) into whichmcTGFβ3 had been transduced, and also a mixture (mcBOTH-OG) of mcBMP2-OGand mcTGFβ3-OG at a 1:1 ratio were inoculated into a medium, andco-cultured to induce differentiation into chondrocytes. Chondrogenicpellets were precipitated by centrifugation to form condensates at day5, day 10, day 20, and day 30 after initiation of differentiationinduction of the OG cells transduced with the minicircle vectors inchondrogenic differentiation medium. Then, for the condensates, cellmorphology, expression of intracellular RFP, and expression level of GFPwere checked.

As a result, as illustrated in FIGS. 13A-13E, it was first identifiedthat the amount of the condensed cells in mcTGFβ3-OG is somewhat lowerthan that in the other experimental groups (FIG. 13A). From 20 daysafter initiation of differentiation induction, the size of the cellcondensate in mcTGFβ3-OG was increased as compared with mcBMP2-OG andmcBOTH-OG, and mcTGFβ3-OG exhibited the cell proliferation rate at asignificantly higher level than that in the other experimental groups(FIG. 13D).

In a case where the expression level of RFP is checked, it wasidentified that the expression level of RFP in the chondrogenic pelletsincreases from 5 days after initiation of differentiation induction inall experimental groups (FIG. 13B). Thereafter, it was identified thatmorphology of the agglutinated cells continues to remain even at day 10(FIG. 13C). The expression of RFP in all experimental groups ofmcBMP2-OG, mcTGFβ3-OG, and mcBOTH-OG tended to increase continuously upto 20 days after initiation of differentiation induction (FIG. 13D). Theexpression tended to decrease from 30 days after initiation ofdifferentiation induction (FIG. 13E). In a case of the OG cell control(NA) transduced with mcMock, it was identified that the expression ofRFP is continuously increased after initiation of differentiation and iskept even after 30 days.

<10-3> Identification of Differentiation Efficiency of ChondrogenicPellet Produced by Differentiation Induction Using mcBMP2 and mcTGF03

Subsequently, in order to analyze characteristics of the differentiatedchondrogenic pellet, expression levels of the marker genes inchondrocyte, which are SOX9, ACAN, COL2A1, COL1A1, and COL10A1, werechecked. In addition, osteoclastogenic capacity of the chondrogenicpellet was identified by checking the expression level of RUNX2, anosteogenic marker. In order to compare differentiation efficiency usingthe minicircle vectors of the present invention with the conventionaltechnique, the expression of the same marker genes was checked for thepositive control (Both rhGF) which had been induced to differentiateinto chondrocytes in a medium containing both the BMP2 and TGFβ3 growthfactors without transduction of the minicircle vectors.

As a result, as illustrated in FIGS. 14A-14D, it was identified that thechondrocyte marker genes are expressed at high levels in allexperimental groups of mcBMP2-OG, mcTGFβ3-OG, and mcBOTH-OG as comparedwith controls of hiPSCs, OG cells, and mcMock-transduced OG cells. Itwas identified that SOX9, which is a marker expressed in earlychondrocytes, is expressed at a significant level in the chondrogenicpellet derived from the cells transduced with the minicircle vectors(FIG. 14A). In a case of ACAN and COL10A1 markers, it was identifiedthat absolute expression levels thereof are observed at lower levelsthan SOX9, and that these markers are expressed at higher levels thanthe positive control, Both rhGF group (FIGS. 14D and 14E). In addition,it was identified that COL2A1 and COL1A1 are also expressed atsignificant levels in mcBMP2-OG, mcTGFβ3-OG, and mcBOTH-OG, and that thechondrocyte marker genes are expressed at significantly higher levelsthan the positive control, Both rhGF group (FIGS. 14C and 14D). On thecontrary, it was identified that the osteogenic marker RUNX2 isexpressed at a lower level than a case where recombinant growth factorsare added, and that RUNX2 is expressed at the lowest level in mcBOTH-OG.

The accumulation level of the extracellular matrix (ECM) was identifiedtogether with the expression of the chondrocyte marker genes. The ECMaccumulation was identified by carrying out alcian blue staining,safranin O staining, and toluidine blue staining for the chondrogenicpellets produced by differentiation induction from mcBMP2-OG,mcTGFβ3-OG, and mcBOTH-OG.

The experimental procedure for this is as follows. Cells of thechondrogenic pellet were first washed with phosphate-buffered saline(PBS). The washed sample was fixed by treatment with 4% paraformaldehydeat room temperature for 2 hours. After the fixation, dehydration wasperformed using an ethanol solution, and washing was performed againusing an ethanol-zylene mixed solution. The washed sample was embeddedin paraffin overnight. The obtained paraffin block was fixed and cutinto 7 μm sections using a microtome to make sample sections. Prior tostaining the respective sections, the sections were placed in an oven at60° C. for at least 10 minutes to raise the temperature. The sectionswere immediately deparaffinized with zylene, hydrated with decreasingethanol concentration, and then rinsed with running tap water for 1minute.

For the alcian blue staining, the sections were immersed in 1% alcianblue solution (Sigma Aldrich, St. Louis, MO, USA) and incubated at roomtemperature for 30 minutes. After the incubation, the sections werewashed again with tap water, counter-stained using nuclear fast redsolution, and then observed with a microscope.

For the safranin O staining, the sections were treated with a solutionof Weigert's hematoxylin (Sigma Aldrich), stained at room temperaturefor 10 minutes, and then washed again with running tap water for 10minutes. The washed sections were stained again with 0.001% Fast Greensolution (Sigma Aldrich) and 0.1% safranin O solution (Sigma Aldrich)for 5 minutes each, and observed with a microscope.

The toluidine blue staining was carried out by immersing dehydratedsections in 0.04% toluidine blue solution and performing incubation for10 minutes. The stained sections were washed with running tap water anddried for 10 minutes until complete drying was achieved. At the end ofthe staining process, the sections were dehydrated with treatment withincreasing ethanol concentration. Ethanol was removed by performingtreatment with 100% zylene twice, mounted on the VectaMount™ PermanentMounting Medium (Vector Laboratories, CA, USA), and then observed with amicroscope.

In addition, in order to identify the types of collagen constituting theECM produced, collagen formation of the chondrogenic pellet was checkedby immunochemical staining, through type 1 collagen and type 2 collagenstaining. Prior to staining the respective sections, the sections wereplaced in an oven at 60° C. for at least 10 minutes to raise thetemperature. The sections were immediately deparaffinized with zylene,hydrated with decreasing ethanol concentration, and then rinsed withrunning tap water for 1 minute.

Then, the sections were immersed in boiling citrate buffer andrehydrated to unmask antigen proteins. The sections after completion ofantigen unmaking were cooled, and then treated with 3% hydrogen peroxidesolution to block activity of peroxidase expressed in tissues. Then, thesections were washed again and blocked with TBS containing 1% BSA.Primary antibodies were diluted with the blocking solution and used. Thesections were treated with anti-type 1 collagen antibodies (1/200dilution; Abcam) or anti-type 2 collagen antibodies (1/100 dilution;Abcam) and incubation therewith was performed at 4° C. overnight. Nextday, the sections were washed with TBS containing 0.1% Tween-20, andtreatment with secondary antibodies was performed. Treatment with thesecondary antibodies (1/200 dilution; Vector Laboratories) was performedat room temperature for 40 minutes and then washing was performed. Afterthe washing, treatment with ABC reagent drops (Vector Laboratories) wasperformed for 30 minutes. Then, the sections were immersed in a DABsolution and incubated for 5 minutes. Counter staining was performed bytreatment with Mayer's hematoxylin (Sigma Aldrich) for 1 minute. Thecounter-stained sections were mounted and microscopically observed withbright illumination.

As a result, as illustrated in FIG. 15 , it was identified that ECM issignificantly formed in all experimental groups. Among these, it wasidentified that overall uniform ECM expression is observed in themcBMP2-OG-derived chondrogenic pellet exhibits, while ECM is expressedin the form of being accumulated in some regions in themcTGFβ3-OG-derived chondrogenic pellet (FIGS. 15A to 15C).

Collagen that constitutes cartilage in vivo may include type 1 collagenand type 2 collagen. It is known that type 1 collagen forms fibrouscartilage and type 2 collagen forms hyaline cartilage. It was identifiedthat collagen is significantly expressed in all experimental groups.However, it was identified that both type 1 collagen and type 2 collagenare observed at high levels in the mcBOTH-OG-derived chondrogenic pellet(FIGS. 15D and 15E). Accordingly, the present inventors expected that inthe course of inducing differentiation into chondrocytes using theminicircle vectors of the present invention, a more effective inductionof differentiation into chondrocytes is possible in a case wheremcBMP2-OG and mcTGFβ3-OG are cultured together for differentiation intochondrocytes.

[Example 11] Identification of Cartilage Regeneration Capacity In Vivoof Chondrogenic Pellet Produced by Differentiation Induction withTransduction of Minicircle Vectors

In order to identify whether the chondrogenic pellet produced bydifferentiation induction using the method of the present inventionactually exhibits cartilage regeneration capacity in vivo in aneffective manner, a regeneration effect was identified by transplanting,into a cartilage defective mouse model, the chondrogenic pellet producedby differentiation induction from mcBOTH-OG.

First, a model mouse having osteochondral defect was produced (FIG.16A). For this purpose, an experimental mouse (Sprague-Dawley) wasanesthetized, and osteochondral defect having a size of 1.5 mm×1.5mm×1.5 mm was induced using a microdrill at articular cartilage of thetrochlear groove of the distal femur. Then, the chondrogenic pellet,produced by differentiation induction for 10 days in <Example 8>, wastransplanted into the osteochondral defect site. The skin on thesurgical site which had been opened for arthrotomy was sealed with anylon thread. The animal model was fed with sufficient feed and drinkingwater for 4 weeks after the surgery. 4 weeks later, the mice weresacrificed, and the corresponding osteochondral defect site was observedby immunohistological staining. For cartilage regeneration effects,degrees of regeneration were checked using the recovery scoresestablished by the International Cartilage Repair Society (ICRS) andcompared.

As a result, as illustrated in FIGS. 16B-16D, it was identified, throughalcian blue staining, toluidine blue staining, and safranin O staining,that ECM formation is induced by the transplanted chondrocytes at thesite where osteochondral defect has been induced. It was identified thatin the control (defect only) in which the defect has been kept for 4weeks and the control into which mcMock-OG-derived chondrocytes havebeen transplanted, ECM accumulation is not observed and the empty spaceis increased. This was identified as indicating that although the cellsof mcMock-OG are transplanted, significant differentiation intochondrocytes does not proceed due to lack of growth factors, and thuscell death is induced.

On the contrary, it was identified that in the experimental group intowhich the mcBOTH-OG-derived chondrogenic pellet has been transplanted,chondrocytes are densely clustered in various regions and produced bydifferentiation induction, and thus a cartilage regeneration effect issignificantly exhibited to induce ECM accumulation (FIGS. 16B to 16D).In addition, it was identified that even in a case where thehistological score is checked, the score is remarkably higher than thecontrol, indicating that cartilage regeneration is effectively achieved(FIG. 16E).

[Example 12] Induction of Differentiation into Chondrocytes withIsolation of Cells by Sizes

In order to establish a method capable of producing chondrocytes of celltherapeutic grade, the present inventors have constructed a method inwhich embryoid bodies (EBs) are produced from induced pluripotent stemcells (iPSCs), mesenchymal-like outgrowth cells (OG cells) are obtained,and the OG cells obtained by size classification through centrifugationare induced to differentiate into chondrocytes (FIG. 17 ).

i) Preparation of iPSCs

First, cord blood mononuclear cell (CBMC)-derived induced pluripotentstem cells were prepared. Here, the CBMCs used were acquired from theCord Blood Bank at Seoul ST. Mary's Hospital, Korea. The cord blood wasdiluted with phosphate buffered saline (PBS) and centrifuged at 850×gfor 30 minutes through a Ficoll gradient to collect CBMCs. Then, theCBMCs were washed and frozen, and kept until use. The CBMCs were thawedimmediately before use, and then resuspended in StemSpan medium(STEMCELL Technological, Vancouver, British Columbia, Canada)supplemented with CC110 cytokine cocktail (STEMCELL). The resultant wascultured for 5 days in a 5% CO₂ incubator at 37° C.

Then, in order to produce iPSCs from the CBMCs, the CBMCs wereinoculated into a 24-well plate at a concentration of 3×10⁵, andreprogramming was induced using the CytoTune-iPS Sendai Reprogrammingkit according to the protocol provided by the manufacturer. Thus,CBMC-derived iPSCs were obtained.

ii) Induction of Embryoid Bodies (EBs) and Outgrowth Cells (OG Cells)from CBMC-Derived iPSCs

The CBMC-derived iPSCs were resuspended in Aggrewell medium (STEMCELL)and inoculated onto a 100-mm culture plate at a concentration of 2×10⁶cells/well. The inoculated iPSCs were cultured in a 37° C. incubator for24 hours. Next day, the medium was replaced with TeSR-E8 medium, andthen culture was further performed for 6 days to obtain EBs. Theobtained EBs were suspended in DMEM medium containing 20% fetal bovineserum (FBS), and cultured on a gelatin-coated plate for 7 days to induceformation of OG cells.

iii) Isolation of OG Cells by Sizes

The formed OG cells were separated from the gelatin-coated plate andpassed through a cell strainer (Thermo Fisher Scientific) having a sizeof 40 μm to remove cell masses. The cells were isolated into single unitcells. The isolated cells were centrifuged so as to be isolated again bysizes. First, the cells were centrifuged at 500 rpm for 5 seconds, andthe precipitated cells were obtained as heavy cells. The supernatant wascentrifuged again at 1,100 rpm for 5 seconds, and the precipitated cellswere obtained as medium cells. In addition, the supernatant wascentrifuged again at 1,500 rpm for 5 seconds, and the precipitated cellswere obtained as light cells.

iv) Induction of Differentiation of OG Cells into Chondrogenic Pellet

The obtained heavy cells, medium cells, and light cells wererespectively counted, inoculated into a chondrogenic differentiationmedium at a concentration of 3×10⁵ cells/tube, and cultured. For thechondrogenic differentiation medium, DMEM medium containing 20% knockoutserum replacement, 1× non-essential amino acid, 1 mM L-glutamine, 1%sodium pyruvate, 1% ITS+Premix, 10⁻⁷ M Dexamethasone, 50 μm ascorbicacid, 40 μg/ml of L-proline, 50 ng/ml of human bone morphogeneticprotein 2, and 10 ng/ml of human transforming growth factor beta 3 wasused. The respective cells were inoculated into the chondrogenicdifferentiation medium, and then centrifuged at 750×g for 5 minutes toprecipitate the cells. Culture was performed at 37° C. for 30 days intotal with daily replacement with fresh medium, so that a finallydifferentiation-induced chondrogenic pellet was obtained.

[Example 13] Identification of Differentiation Induction Markers in OGCells Isolated by Sizes

In order to identify whether distinguished differentiation capacity intochondrocytes is exhibited as OG cells are isolated by sizes,characteristics of the OG cells, which had been isolated by sizesthrough centrifugation, were first identified.

First, cell morphology of the respective heavy OG cells, medium OGcells, and light OG cells obtained in the step iii) of [Example 12] wasobserved with a microscope. As a result, as illustrated in FIGS. 18A and18B, it was identified that the cells are isolated depending on theirsizes.

Then, the respective cells were equally counted to 5×10⁵ cells for eachsample. Then, the respective cells were disrupted using Trizol (LifeTechnologies), and mRNA was extracted therefrom. Using the mRNA as atemplate, cDNA was synthesized using the RevertAid™ First Strand cDNAsynthesis kit (Thermo Fisher Scientific) according to the protocolprovided by the manufacturer. Using the synthesized cDNA as a template,PCR was carried out again using the primer sequences listed in [Table 9]below, to identify mRNA expression levels of SOX9, which is atranscription factor that helps induce differentiation intochondrocytes, and COL10, which is a hypertrophy marker that helps cellgrowth. The mRNA expression levels were identified by electrophoresis,and then relative expression levels of intracellular SOX9 and COL10A1were analyzed based on GAPDH for quantitative analysis.

As a result, as illustrated in FIGS. 18C to 18E, it was identified thatthe heavy cells exhibit a low expression level of SOX9 and a highexpression level of COL10, and the light cells exhibit a relatively highexpression level of SOX9 and no expression of COL10.

TABLE 9 List of primers used for identifying expression of markers in inducing differentiation into chondrocytes depending on sizesof OG cells and sequences therefor Target Name Direction Primer SequenceSize SOX5 Forward CAGCCAGAGTTAGCACAATAGG 104 ReverseCTGTTGTTCCCGTCGGAGTT SOX6 Forward GGATGCAATGCCCAGGATTT 141 ReverseTGAATGGTACTGACAAGTGTTG G SOX9 Forward GAACGCACATCAAGACGGAG 631 ReverseTCTCGTTGATTTCGCTGCTC COL2A1 Forward GGCAATAGCAGGTTCACGTACA  79 ReverseCGATAACAGTCTTGCCCCACTT A COL1A1 Forward TCTGCGACAACGGCAAGGTG 146 ReverseGACGCCGGTGGTTTCTTGGT COL10A1 Forward GTCTGCTTTTACTGTTATTCTCT 108 CCAAAReverse TGCTGTTGCCTGTTATACAAAA TTTT ACAN Forward AGCCTGCGCTCCAATGACT 107Reverse TAATGGAACACGATGCCTTTCA CHAD Forward GATCCCCAAGGTGTCAGAGAAG  66Reverse GCCAGCACCGGGAAGTT PRG4 Forward AAAGTCAGCACATCTCCCAA 108 ReverseGTGTCTCTTTAGCGGAAGTAGT C RUNX2 Forward TCTTAGAACAAATTCTGCCCTTT 136Reverse TGCTTTGGTCTTGAAATCACA OPN Forward GGGAGTACGAATACACGGGC  92Reverse TCGGTAATTGTCCCCACGAG BGLAP Forward ATGAGAGCCCTCACACTCCT 117Reverse CTTGGACACAAAGGCTGCAC

In order to identify again the expression of SOX9 and COL10A1 at theprotein level, fluorescence immunoassay was performed. The respectivecells were counted to the same number, then washed with PBS, and fixedwith 4% paraformaldehyde. The fixed cells were treated with 0.1% TritonX-100 for 10 minutes to have permeability, and then precipitated toobtain cells. The cells were blocked at room temperature for 30 minutesby treatment with PBS (PBA) containing 2% bovine serum albumin (BSA).The blocked cells were treated with anti-SOX9 antibodies or anti-CTLA10antibodies as primary antibodies, and then allowed to react at roomtemperature for 2 hours. Then, Alexa Fluor 594-antibodies were addedthereto, and reaction was induced in the dark for 1 hour. Aftercompletion of the reaction, the cells were washed with PBA, and thestained cells were observed with a fluorescence microscope. Nuclei ofthe cells were stained with DAPI.

As a result, as illustrated in FIGS. 18F and 18G, it was identified thatthe heavy OG cells exhibit a low expression level of SOX9 and a highexpression level of COL10, and the light OG cells exhibit a relativelyhigh expression level of SOX9 and no expression of COL10. From this, itwas identified that a consistent pattern is exhibited between theresults obtained at the mRNA expression level and the results obtainedat the protein level.

[Example 14] Characterization of OG Cell-Derived Chondrogenic Pellet

<14-1> Identification of Structure Formation Capacity for ChondrogenicPellet Depending on Sizes of OG Cells

It was identified whether among the chondrogenic pellets differentiatedthrough the method of the present invention, a degree of differentiationvaries depending on sizes of the OG cells.

Specifically, the colony of the chondrogenic pellet produced bydifferentiation induction in the step iv) of [Example 12] was fixed with4% paraformaldehyde at room temperature for 2 hours, and dehydrated witha solution obtained by mixing ethanol and zylene. The dehydrated pelletwas fixed with paraffin, and then cut into 7 μm sections to preparesections. For the prepared sections, toluidine blue staining, safranin Ostaining, and alcian blue staining were carried out. Each sample waschecked with a microscope.

As a result, as illustrated in FIGS. 19A-19B, it was identified that theOG cells isolated depending on their sizes also exhibit different sizesof colonies as the time for inducing differentiation into chondrogenicpellet has elapsed (FIG. 19A). After completion of differentiationinduction, in a case where the chondrogenic pellets are stained andhistologically checked, it was identified that the chondrogenic pelletproduced by differentiation induction from the light cells has a morestable structure (FIG. 19B). On the contrary, it was identified that thechondrogenic pellet produced by differentiation induction from the heavycells has a loose structure in terms of cartilaginous tissue, indicatingsimilar characteristics to those of osteoarthritic patients.

<14-2> Identification of Difference in Expression Levels of Markers inChondrogenic Pellet Depending on Sizes of OG Cells

In order to identify whether chondrocyte marker genes are expressed inthe chondrogenic pellet differentiated through the method of the presentinvention, gene expression levels of the chondrogenic differentiationpromotion markers SOX5, SOX6, and SOX9; the collagen markers COL2A1,COL1A1, and COL10A10; the extracellular matrix proteins ACAN, CHAD, andPRG4; and the bone differentiation markers RUNX2, OPN, and BGLAP werechecked.

Specifically, the chondrogenic pellet produced by differentiationinduction in the step iv) of [Example 12] was obtained, rapidly frozenwith liquid nitrogen, and then ground with pestle and mortar. To eachground sample was added Trizol so that the chondrogenic pellet wasdisrupted, and total mRNA was extracted therefrom. Using the extractedmRNA as a template, cDNA was synthesized. Using the cDNA again as atemplate, PCR was performed with the primer sequences shown in [Table 9]above, to identify mRNA expression levels of the respective markers. Forthe mRNA expression levels, relative expression levels were comparedbased on GAPDH. Thus, in the differentiated chondrogenic pellets,relative expression levels of the marker genes depending on the sizes ofthe OG cells were analyzed.

As a result, as illustrated in FIG. 20 , it was identified that as theOG cells are smaller in size, high gene expression of the extracellularmatrix proteins is observed in the chondrogenic pellet produced bydifferentiation induction therefrom, while significance for thechondrogenic differentiation markers is decreased among the groups. Fromthis, it was identified that the light OG cell-derived chondrogenicpellet can significantly exhibit formation of hyaline cartilage and thushas high value.

[Example 15] Identification of Cause by which Different Capacity ofDifferentiating into Chondrogenic Pellet is Exhibited Depending on Sizesof OG Cells

It was identified that OG cells exhibit different differentiationcapacities of differentiating into chondrogenic pellets depending ontheir sizes, and that as the OG cells are smaller, better capacity ofdifferentiating into chondrogenic pellet is exhibited. Thus, it wasintended to find a factor that prevents heavy OG cells from exhibiting asignificant differentiation capacity.

Specifically, total mRNA was extracted, respectively, from the heavy OGcells, the medium OG cells, and the light OG cells obtained in the stepiii) of [Example 12], and then the gene expression level thereof wasidentified by microarray analysis. From the results of microarrayanalysis, the expression levels of genes in the medium OG cells and thelight OG cells were compared to expression levels of genes in the heavyOG cells, in which a gene of which the expression level is specificallychanged in the heavy OG cells was screened.

As a result, as shown in [Table 10] and [Table 11] below, it wasidentified that as the OG cells are larger, the expression level ofinsulin-like growth factor 2 (IGF2) is remarkably higher.

TABLE 10 Fold Change Gene Accession Gene Symbol Gene Name of H/M, LUpregulated NM_000612 IGF2 Insulin-like Growth Factor 2 97.48 NM_181501ITGA1 Integrin, Alpha 1 21.62 NM_002398 MEIS1 Meis Homeobox 1 18.70NM_005994 TBX2 T-box 2 11.89 NM_177963 SYT12 Synaptotagmin XII 11.60NM_001083 PDE5A Phosphodiesterase 5A, cGMP-specific 10.22 NM_021110COL14A1 Collagen, Type XIV, Alpha 1 9.73 NM_130385 MRVI1 MurineRetrovirus Integration Site 1 Homolog 8.71 NM_152864 NKAIN4Na—K-Transporting ATPase Interacting 4 8.50 NM_001452 FOXF2 Forkhead BoxF2 7.78 NM_001257995 LMO7DN LMO7 Downstream Neighbor 7.60 NM_005940MMP11 Matrix Metallopeptidase 11 7.51 NM_001290268 FAM65C Family withSequence Similarity 65, Member C 7.33 NM_018440 PAG1 PhosphoproteinMembrane Anchor with 7.26 Glycosphingolipid Microdomains 1 NM_001958EEF1A2 Eukaryotic Translation Elongation Factor 1 Alpha 2 7.03 NM_004572PKP2 Plakophilin 2 6.85 NM_001031804 MAF V-Maf Avian MusculoaponeuroticFibrosarcoma 6.75 Oncogene Homolog NM_001993 F3 Coagulation factor III6.63 NM_001271948 PPP2R2B Protein Phosphatase 2, Regulatory Subunit B,Beta 6.56 NM_001105521 JAKMIP3 Janus Kinase and Microtubule InteractingProtein 3 6.48 NM_022166 XYLT1 Xylosyltransferase I 6.33 NM_000399 EGR2Early Growth Response 2 6.17 NM_023037 FRY Furry Homolog (Drosophila)6.12 NM_002653 PITX1 Paired-like Homeodomain 1 5.91 NM_006308 HSPB3 HeatShock 27 kDa protein 3 5.87 NR_125749 TBX2-AS1 TBX2 Antisense RNA 1 5.78NM_000955 PTGER1 Prostaglandin E Receptor 1 (Subtype EP1) 5.78 NM_004155SERPINB9 Serpin Peptidase Inhibitor, Clade B, Member 9 5.76 NR_034095LINC01197 Long Intergenic Non-protein Coding RNA 1197 5.75 NM_001845COL4A1 Collagen, Type IV, Alpha 1 5.65

TABLE 11 Fold Change Gene Accession Gene Symbol Gene Name of H/M, LDownregulated NM_015429 ABI3BP ABI Family, Member 3 (NESH) BindingProtein −22.20 NM_001007156 NTRK3 Neurotrophic Tyrosine Kinase,Receptor, Type 3 −18.56 NM_024893 SYNDIG1 Synapse DifferentiationInducing 1 −17.16 NM_020311 ACKR3 Atypical Chemokine Receptor 3 −15.89NM_017680 ASPN Asporin −14.44 NM_001252 CD70 CD70 Molecule −14.25NM_003485 GPR68 G Protein-coupled Receptor 68 −13.55 NM_006100 ST3GAL6ST3 Beta-galactoside Alpha-2,3-sialyltransferase 6 −11.77 NR_038236LINC00968 Long Intergenic Non-protein Coding RNA 968 −11.36 NM_003836DLK1 Delta-like 1 Homolog (Drosophila) −10.64 NR_102279 HOXB-AS1 HOXBCluster Antisense RNA 1 −10.03 NM_001252065 SYT7 Synaptotagmin VII −9.92NM_013363 PCOLCE2 Procollagen C-endopeptidase Enhancer 2 −9.54 NM_005099ADAMTS4 ADAM Metallopeptidase with Thrombospondin Type 1 −9.44 Motif, 4NM_015225 PRUNE2 Prune Homolog 2 (Drosophila) −9.36 NM_005202 COL8A2Collagen, Type VIII, Alpha 2 −9.09 NM_000963 PTGS2Prostaglandin-endoperoxide Synthase 2 −9.06 NM_032528 ST6GAL2 ST6Beta-galactosamide alpha-2,6-sialyltranferase 2 −8.87 NM_006208 ENPP1Ectonucleotide Pyrophosphatase/Phosphodiesterase 1 −8.55 NM_005949 MT1FMetallothionein 1F −8.47 NM_000337 SGCD Sarcoglyan, Delta −8.07NM_007281 SCRG1 Stimulator of Chondrogenesis 1 −7.93 NM_024119 DHX58DEXH (Asp-Glu-X-His) Box Polypeptide 58 −7.42 NM_032849 MEDAG MesentericEstrogen-dependent Adipogenesis −7.24 NM_198449 EMB Embigin −7.10NM_002245 KCNK1 Potassium Channel, Two Pore Domain Subfamily K, −7.07Member 1 NM_130783 TSPAN18 Tetraspanin 18 −6.93 NM_001146037 SLC14A1Solute Carrier Family 14, Member 1 −6.78 NM_020386 HRASLS HRAS-likeSuppressor −6.72 NM_003480 MFAP5 Microfibrillar Associated Protein 5−6.54 H: Heavy OG; M: Medium OG; L: Light OG

[Example 16] Identification of Differentiation Capacity of ChondrogenicPellet Produced by Differentiation Induction in Environment Treated withIGF2 Inhibitor

<16-1> Identification of Degree of Proliferation of OG Cells Cultured inIGF2 Inhibitor-Treated Medium

From [Example 15], it was identified that depending on sizes of OGcells, chondrogenic pellets differentiated therefrom exhibit differentexpression levels of IGF2. Thus, it was intended to identify whether theOG cells exhibit changed differentiation capacity in a case wheretreatment with chromeceptin, an IGF2 inhibitor, is performed.

First, among the OG cells obtained in the step iii) of [Example 12], theheavy cells were cultured for 3 days while treating the medium withchromeceptin at a concentration of 2 nM to 2 μM, in the course ofinducing differentiation into a chondrogenic pellet according to thestep iv). Then, morphology of the cells was observed over the treatmenttime, and the number of the proliferated cells was counted.

As a result, as illustrated in FIG. 21 , it was identified that a degreeof proliferation of the cells decreases with increased treatmentconcentration of chromeceptin, and that after 72 hours, the number of OGcells induced to differentiate in the medium containing 2 μMchromeceptin decreases by about 3 times or higher as compared with theuntreated control.

<16-2> Identification of Chondrogenic Differentiation Markers of OGCells Cultured in IGF2 Inhibitor-Treated Medium

It was identified that in a case where treatment with chromeceptin, anIGF2 inhibitor, is performed, proliferative capacity of the OG cellsalso changes with increased concentration of chromeceptin. Thus, it wasintended to identify whether expression levels of chondrogenicdifferentiation markers differ by treatment with chromeceptin.

Accordingly, heavy OG cells were induced to differentiate into achondrogenic pellet while performing treatment with chromeceptin underthe conditions of Example <16-1>, during which the OG cells wereobtained, and mRNA was extracted therefrom and used to synthesize cDNA.Using the synthesized cDNA as a template, expression levels of SOX9,COL2A1, COL10A1, and IGF2 were quantitatively analyzed.

As a result, as illustrated in FIG. 22 , it was first identified thatthe expression level of IGF2 in the OG cells decreases with increasedtreatment concentration of chromeceptin. On the contrary, it wasidentified that the expression level of SOX9, a chondrogenicdifferentiation marker, significantly increases with increased treatmentconcentration of chromeceptin, and that the expression level of SOX9remarkably increases in a cell sample which has been treated with 2 μMchromeceptin as compared with the untreated control (Normal Ctrl).

<16-3> Identification of Chondrocyte Markers in Chondrogenic PelletProduced by Differentiation Induction in IGF2 Inhibitor-Treated Medium

It was identified that the expression levels of chondrogenicdifferentiation markers can significantly increase even in heavy OGcells by treatment with an IGF2 inhibitor. Accordingly, it was intendedto identify whether significant differentiation has occurred in thechondrogenic pellet produced by differentiation induction in thisenvironment.

First, among the OG cells obtained in the step iii) of [Example 12], theheavy cells were cultured for 3 days while treating the medium withchromeceptin at a concentration of 2 mM, in the course of inducingdifferentiation into a chondrogenic pellet according to the step iv).Experimental groups were divided into two groups, that is, theexperimental group (before aggregation) in which differentiation intochondrocytes had been induced by performing treatment with chromeceptinfrom the beginning of formation of a chondrogenic pellet, and theexperimental group (after aggregation) in which differentiation intochondrocytes had been induced by performing treatment with chromeceptinfrom 7 days after induction of formation of a chondrogenic pellet. Thedifferentiated chondrogenic pellets were respectively obtainedtherefrom. Then, mRNA was extracted from each of the obtainedchondrogenic pellets, and the expression levels of COL2A1, SOX9, COL1A1and COL10A1 were checked.

As a result, as illustrated in FIGS. 23A-23B, it was identified that asdifferentiation into a chondrogenic pellet is induced with treatmentwith chromeceptin, the expression level of SOX9, a chondrocyte marker,is significantly increased in a case of being at a later stage (afteraggregation) of formation of the chondrogenic pellet as compared with acase of being an initial stage (before aggregation) of formation of thechondrogenic pellet.

1. A method for producing chondrocytes obtained by differentiationinduction from stem cells, comprising: i) culturing induced pluripotentstem cells (iPSCs) to generate embryoid bodies (EBs); ii) culturing theEBs generated in step i) in a gelatin-coated medium, to obtain outgrowthcells (OG cells); iii) transducing the OG cells obtained in step ii)with either or both of a minicircle vector that contains a base sequenceencoding BMP2 and a minicircle vector that contains a base sequenceencoding TGFβ3; iv) inducing differentiation of the OG cells transducedin step iii) into chondrocytes; and v) obtaining the chondrocytesproduced by differentiation induction in step iv).
 2. A method forproducing chondrocytes obtained by differentiation induction from stemcells, comprising: i) culturing iPSCs to generate EBs; ii) culturing theEBs generated in step i) in a gelatin-coated medium, to obtain OG cells;iii) transducing the OG cells obtained in step ii) with a minicirclevector that contains a base sequence encoding BMP2; iv) transducing theOG cells obtained in step ii) with a minicircle vector that contains abase sequence encoding TGFβ3; v) performing mixed culture of the OGcells transduced in step iii) and the OG cells transduced in step iv),so that the OG cells are induced to differentiate into chondrocytes; andvi) obtaining the chondrocytes produced by differentiation induction instep v).
 3. The method according to claim 1, wherein the minicirclevector that contains a base sequence encoding BMP2 is a non-viralvector, the non-viral vector, (a) containing a gene expression cassettethat contains a CMV promoter, a BMP2 gene consisting of the basesequence of SEQ ID NO: 1, and an SV40 polyadenylation sequence; (b)containing the att attachment sequence of bacteriophage lambda, locatedoutside the gene expression cassette of (a); and (c) not containing areplication origin and an antibiotic resistance gene.
 4. The methodaccording to claim 1, wherein the minicircle vector that contains a basesequence encoding TGFβ3 is a non-viral vector, the non-viral vector, (a)containing a gene expression cassette that contains a CMV promoter, aTGFβ3 gene consisting of the base sequence of SEQ ID NO: 2, and an SV40polyadenylation sequence; (b) containing the att attachment sequence ofbacteriophage lambda, located outside the gene expression cassette of(a); and (c) not containing a replication origin and an antibioticresistance gene.
 5. The method according to claim 1, wherein the step ofinducing differentiation of the OG cells into chondrocytes is performedby culturing the OG cells in a medium containing no recombinant growthfactor for 3 to 30 days.
 6. A chondrocyte produced by the method ofclaim
 1. 7. A chondrocyte produced by the method of claim
 2. 8. Themethod according to claim 2, wherein the minicircle vector that containsa base sequence encoding BMP2 is a non-viral vector, the non-viralvector, (a) containing a gene expression cassette that contains a CMVpromoter, a BMP2 gene consisting of the base sequence of SEQ ID NO: 1,and an SV40 polyadenylation sequence; (b) containing the att attachmentsequence of bacteriophage lambda, located outside the gene expressioncassette of (a); and (c) not containing a replication origin and anantibiotic resistance gene.
 9. The method according to claim 2, whereinthe minicircle vector that contains a base sequence encoding TGFβ3 is anon-viral vector, the non-viral vector, (a) containing a gene expressioncassette that contains a CMV promoter, a TGFβ3 gene consisting of thebase sequence of SEQ ID NO: 2, and an SV40 polyadenylation sequence; (b)containing the att attachment sequence of bacteriophage lambda, locatedoutside the gene expression cassette of (a); and (c) not containing areplication origin and an antibiotic resistance gene.
 10. The methodaccording to claim 2, wherein the step of inducing differentiation ofthe OG cells into chondrocytes is performed by culturing the OG cells ina medium containing no recombinant growth factor for 3 to 30 days.